Patched genes and uses related thereto

ABSTRACT

Invertebrate and vertebrate patched genes are provided, including the mouse and human patched genes, as well as methods for isolation of related genes, where the genes may be of different species or in the same family. Having the ability to regulate the expression of the patched gene, allows for the elucidation of embryonic development, cellular regulation associated with signal transduction by the patched gene, the identification of agonist and antagonist to signal transduction, identification of ligands for binding to patched, isolation of the ligands, and assaying for levels of transcription and expression of the patched gene.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser. No. 08/319,745, the disclosure of which is herein incorporated by reference.

INTRODUCTION

[0002] 1. Technical Field

[0003] The field of this invention concerns segment polarity genes and their uses.

[0004] 2. Background

[0005] Segment polarity genes were discovered in flies as mutations which change the pattern of structures of the body segments. Mutations in the genes cause animals to develop the changed patterns on the surfaces of body segments, the changes affecting the pattern along the head to tail axis. For example, mutations in the gene patched cause each body segment to develop without the normal structures in the center of each segment. In their stead is a mirror image of the pattern normally found in the anterior segment. Thus cells in the center of the segment make the wrong structures, and point them in the wrong direction with reference to the over all head-to-tail polarity of the animal. About sixteen genes in the class are known. The encoded proteins include kinases, transcription factors, a cell junction protein, two secreted proteins called wingless (WG) and hedgehog (HH), a single transmembrane protein called patched (PTC), and some novel proteins not related to any known protein. All of these proteins are believed to work together in signaling pathways that inform cells about their neighbors in order to set cell fates and polarities.

[0006] Many of the segment polarity proteins of Drosophila and other invertebrates are closely related to vertebrate proteins, implying that the molecular mechanisms involved are ancient. Among the vertebrate proteins related to the fly genes are En-1 and -2, which act in vertebrate brain development and WNT-1, which is also involved in brain development, but was first found as the oncogene implicated in many cases of mouse breast cancer. In flies, the patched gene is transcribed into RNA in a complex and dynamic pattern in embryos, including fine transverse stripes in each body segment primordium. The encoded protein is predicted to contain many transmembrane domains. It has no significant similarity to any other known protein. Other proteins having large numbers of transmembrane domains include a variety of membrane receptors, channels through membranes and transporters through membranes.

[0007] The hedgehog (HH) protein of flies has been shown to have at least three vertebrate relatives: Sonic hedgehog (Shh); Indian hedgehog, and Desert hedgehog. The Shh is expressed in a group of cells at the posterior of each developing limb bud. This is exactly the same group of cells found to have an important role in signaling polarity to the developing limb. The signal appears to be graded, with cells close to the posterior source of the signal forming posterior digits and other limb structures and cells farther from the signal source forming more anterior structures. It has been known for many years that transplantation of the signaling cells, a region of the limb bud known as the “zone of polarizing activity (ZPA)” has dramatic effects on limb patterning. Implanting a second ZPA anterior to the limb bud causes a limb to develop with posterior features replacing the anterior ones (in essence little fingers instead of thumbs). Shh has been found to be the long sought ZPA signal. Cultured cells making Shh protein (SHH), when implanted into the anterior limb bud region, have the same effect as an implanted ZPA. This establishes that Shh is clearly a critical trigger of posterior limb development.

[0008] The factor in the ZPA has been thought for some time to be related to another important developmental signal that polarizes the developing spinal cord. The notochord, a rod of mesoderm that runs along the dorsal side of early vertebrate embryos, is a signal source that polarizes the neural tube along the dorsal-ventral axis. The signal causes the part of the neural tube nearest to the notochord to form floor plate, a morphologically distinct part of the neural tube. The floor plate, in turn, sends out signals to the more dorsal parts of the neural tube to further determine cell fates. The ZPA was reported to have the same signaling effect as the notochord when transplanted to be adjacent to the neural tube, suggesting the ZPA makes the same signal as the notochord. In keeping with this view, Shh was found to be produced by notochord cells and floor plate cells. Tests of extra expression of Shh in mice led to the finding of extra expression of floor plate genes in cells which would not normally turn them on. Therefore Shh appears to be a component of the signal from notochord to floor plate and from floor plate to more dorsal parts of the neural tube. Besides limb and neural tubes, vertebrate hedgehog genes are also expressed in many other tissues including, but not limited to the peripheral nervous system, brain, lung, liver, kidney, tooth primordia, genitalia, and hindgut and foregut endoderm.

[0009] PTC has been proposed as a receptor for HH protein based on genetic experiments in flies. A model for the relationship is that PTC acts through a largely unknown pathway to inactivate both its own transcription and the transcription of the wingless segment polarity gene. This model proposes that HH protein, secreted from adjacent cells, binds to the PTC receptor, inactivates it, and thereby prevents PTC from turning off its own transcription or that of wingless. A number of experiments have shown coordinate events between PTC and HH.

[0010] Relevant Literature

[0011] Descriptions of patched, by itself or its role with hedgehog may be found in Hooper and Scott, Cell 59, 751-765 (1989); Nakano et al., Nature, 341, 508-513 (1989) (both of which also describes the sequence for Drosophila patched) Simcox et al., Development 107, 715-722 (1989); Hidalgo and Ingham, Development, 110, 291-301 (1990); Phillips et al., Development, 110, 105-114 (1990); Sampedro and Guerrero, Nature 353, 187-190 (1991); Ingham et al., Nature 353, 184-187 (1991); and Taylor et al., Mechanisms of Development 42, 89-96 (1993). Discussions of the role of hedgehog include Riddle et al., Cell 75, 1401-1416 (1993); Echelard et al., Cell 75, 1417-1430 (1993); Krauss et al., Cell 75, 1431-1444 (1993); Tabata and Kornberg, Cell 76, 89-102 (1994); Heemskerk & DiNardo, Cell 76, 449-460 (1994); Relink et al., Cell 76, 761-775 (1994); and a short review article by Ingham, Current Biology 4, 347-350 (1994). The sequence for the Drosophila 5′ non-coding region was reported to the GenBank, accession number M28418, referred to in Hooper and Scott (1989), supra. See also, Forbes, et al., Development 1993 Supplement 115-124.

SUMMARY OF THE INVENTION

[0012] Methods for isolating patched genes, particularly mammalian patched genes, including the mouse and human patched genes, as well as invertebrate patched genes and sequences, are provided. The methods include identification of patched genes from other species, as well as members of the same family of proteins. The subject genes provide methods for producing the patched protein, where the genes and proteins may be used as probes for research, diagnosis, binding of hedgehog protein for its isolation and purification, gene therapy, as well as other utilities.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a graph having a restriction map of about 10 kbp of the 5′ region upstream from the initiation codon of Drosophila patched gene and bar graphs of constructs of truncated portions of the 5′ region joined to β-galactosidase, where the constructs are introduced into fly cell lines for the production of embryos. The expression of β-gal in the embryos is indicated in the right-hand table during early and late development of the embryo. The greater the number of +'s, the more intense the staining.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0014] Methods are provided for identifying members of the patched (ptc) gene family from invertebrate and vertebrate, e.g. mammalian, species, as well as the entire cDNA sequence of the mouse and human patched gene. Also, sequences for invertebrate patched genes are provided. The patched gene encodes a transmembrane protein having a large number of transmembrane sequences.

[0015] In identifying the mouse and human patched genes, primers were employed to move through the evolutionary tree from the known Drosophila ptc sequence. Two primers are employed from the Drosophila sequence with appropriate restriction enzyme linkers to amplify portions of genomic DNA of a related invertebrate, such as mosquito. The sequences are selected from regions which are not likely to diverge over evolutionary time and are of low degeneracy. Conveniently, the regions are the N-terminal proximal sequence, generally within the first 1.5 kb, usually within the first 1 kb, of the coding portion of the cDNA, conveniently in the first hydrophilic loop of the protein. Employing the polymerase chain reaction (PCR) with the primers, a band can be obtained from mosquito genomic DNA. The band may then be amplified and used in turn as a probe. One may use this probe to probe a cDNA library from an organism in a different branch of the evolutionary tree, such as a butterfly. By screening the library and identifying sequences which hybridize to the probe, a portion of the butterfly patched gene may be obtained. One or more of the resulting clones may then be used to rescreen the library to obtain an extended sequence, up to and including the entire coding region, as well as the non-coding 5′- and 3′-sequences. As appropriate, one may sequence all or a portion of the resulting cDNA coding sequence.

[0016] One may then screen a genomic or cDNA library of a species higher in the evolutionary scale with appropriate probes from one or both of the prior sequences. Of particular interest is screening a genomic library, of a distantly related invertebrate, e.g. beetle, where one may use a combination of the sequences obtained from the previous two species, in this case, the Drosophila and the butterfly. By appropriate techniques, one may identify specific clones which bind to the probes, which may then be screened for cross hybridization with each of the probes individually. The resulting fragments may then be amplified, e.g. by subcloning.

[0017] By having all or parts of the 4 different patched genes, in the presently illustrated example, Drosophila (fly), mosquito, butterfly and beetle, one can now compare the patched genes for conserved sequences. Cells from an appropriate mammalian limb bud or other cells expressing patched, such as notochord, neural tube, gut, lung buds, or other tissue, particularly fetal tissue, may be employed for screening. Alternatively, adult tissue which produces patched may be employed for screening. Based on the consensus sequence available from the 4 other species, one can develop probes where at each site at least 2 of the sequences have the same nucleotide and where the site varies that each species has a unique nucleotide, inosine may be used, which binds to all 4 nucleotides.

[0018] Either PCR may be employed using primers or, if desired, a genomic library from an appropriate source may be probed. With PCR, one may use a cDNA library or use reverse transcriptase-PCR (RT-PCR), where mRNA is available from the tissue. Usually, where fetal tissue is employed, one will employ tissue from the first or second trimester, preferably the latter half of the first trimester or the second trimester, depending upon the particular host. The age and source of tissue will depend to a significant degree on the ability to surgically isolate the tissue based on its size, the level of expression of patched in the cells of the tissue, the accessibility of the tissue, the number of cells expressing patched and the like. The amount of tissue available should be large enough so as to provide for a sufficient amount of mRNA to be usefully transcribed and amplified. With mouse tissue, limb bud of from about 10 to 15 dpc (days post conception) may be employed.

[0019] In the primers, the complementary binding sequence will usually be at least 14 nucleotides, preferably at least about 17 nucleotides and usually not more than about 30 nucleotides. The primers may also include a restriction enzyme sequence for isolation and cloning. With RT-PCR, the mRNA may be enriched in accordance with known ways, reverse transcribed, followed by amplification with the appropriate primers. (Procedures employed for molecular cloning may be found in Molecular Cloning: A Laboratory Manual, Sambrook et al., eds., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1988). Particularly, the primers may conveniently come from the N-terminal proximal sequence or other conserved region, such as those sequences where at least five amino acids are conserved out of eight amino acids in three of the four sequences. This is illustrated by the sequences (SEQ ID NO:11) IIPLDCFWEG, (SEQ ID NO:12) LIVGG, and (SEQ ID NO:13) PFFWEQY. Resulting PCR products of expected size are subcloned and may be sequenced if desired.

[0020] The cloned PCR fragment may then be used as a probe to screen a cDNA library of mammalian tissue cells expressing patched, where hybridizing clones may be isolated under appropriate conditions of stringency. Again, the cDNA library should come from tissue which expresses patched, which tissue will come within the limitations previously described. Clones which hybridize may be subcloned and rescreened. The hybridizing subclones may then be isolated and sequenced or may be further analyzed by employing RNA blots and in situ hybridizations in whole and sectioned embryos. Conveniently, a fragment of from about 0.5 to 1 kbp of the N-terminal coding region may be employed for the Northern blot.

[0021] The mammalian gene may be sequenced and as described above, conserved regions identified and used as primers for investigating other species. The N-terminal proximal region, the C-terminal region or an intermediate region may be employed for the sequences, where the sequences will be selected having minimum degeneracy and the desired level of conservation over the probe sequence.

[0022] The DNA sequence encoding PTC may be cDNA or genomic DNA or fragment thereof, particularly complete exons from the genomic DNA, may be isolated as the sequence substantially free of wild-type sequence from the chromosome, may be a 50 kbp fragment or smaller fragment, may be joined to heterologous or foreign DNA, which may be a single nucleotide, oligonucleotide of up to 50 bp, which may be a restriction site or other identifying DNA for use as a primer, probe or the like, or a nucleic acid of greater than 50 bp, where the nucleic acid may be a portion of a cloning or expression vector, comprise the regulatory regions of an expression cassette, or the like. The DNA may be isolated, purified being substantially free of proteins and other nucleic acids, be in solution, or the like.

[0023] The subject gene may be employed for producing all or portions of the patched protein. The subject gene or fragment thereof, generally a fragment of at least 12 bp, usually at least 18 bp, may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host. Fragments will usually be immediately joined at the 5′ and/or 3′ terminus to a nucleotide or sequence not found in the natural or wild-type gene, or joined to a label other than a nucleic acid sequence. For expression, an expression cassette may be employed, providing for a transcriptional and translational initiation region, which may be inducible or constitutive, the coding region under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. Various transcriptional initiation regions may be employed which are functional in the expression host. The peptide may be expressed in prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression. For large production of the protein, a unicellular organism or cells of a higher organism, e.g. eukaryotes such as vertebrates, particularly mammals, may be used as the expression host, such as E. coli, B, subtilis, S. cerevisiae, and the like. In many situations, it may be desirable to express the patched gene in a mammalian host, whereby the patched gene will be transported to the cellular membrane for various studies. The protein has two parts which provide for a total of six transmembrane regions, with a total of six extracellular loops, three for each part. The character of the protein has similarity to a transporter protein. The protein has two conserved glycosylation signal triads.

[0024] The subject nucleic acid sequences may be modified for a number of purposes, particularly where they will be used intracellularly, for example, by being joined to a nucleic acid cleaving agent, e.g. a chelated metal ion, such as iron or chromium for cleavage of the gene; as an antisense sequence; or the like. Modifications may include replacing oxygen of the phosphate esters with sulfur or nitrogen, replacing the phosphate with phosphoramide, etc.

[0025] With the availability of the protein in large amounts by employing an expression host, the protein may be isolated and purified in accordance with conventional ways. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. The purified protein will generally be at least about 80% pure, preferably at least about 90% pure, and may be up to 100% pure. By pure is intended free of other proteins, as well as cellular debris.

[0026] The polypeptide may be used for the production of antibodies, where short fragments provide for antibodies specific for the particular polypeptide, whereas larger fragments or the entire gene allow for the production of antibodies over the surface of the polypeptide or protein, where the protein may be in its natural conformation.

[0027] Antibodies may be prepared in accordance with conventional ways, where the expressed polypeptide or protein may be used as an immunogen, by itself or conjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg, other viral or eukaryotic proteins, or the like. Various adjuvants may be employed, with a series of injections, as appropriate. For monoclonal antibodies, after one or more booster injections, the spleen may be isolated, the splenocytes immortalized, and then screened for high affinity antibody binding. The immortalized cells, e.g. hybridomas, producing the desired antibodies may then be expanded. For further description, see Monoclonal Antibodies: A Laboratory Manual, Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1988. If desired, the mRNA encoding the heavy and light chains may be isolated and mutigenized by cloning in E. coli, and the heavy and light chains may be mixed to further enhance the affinity of the antibody. The antibodies may find use in diagnostic assays for detection of the presence of the PTC protein on the surface of cells or to inhibit the transduction of signal by the PTC protein ligand by competing for the binding site.

[0028] The mouse patched gene (SEQ ID NO:09) encodes a protein (SEQ ID NO:10) which has about 38% identical amino acids to fly PTC (SEQ ID NO:6) over about 1,200 amino acids. This amount of conservation is dispersed through much of the protein excepting the C-terminal region. The mouse protein also has a 50 amino acid insert relative to the fly protein. The human patched gene (SEQ ID NO:18) contains an open reading fram of about 1450 amino acids (SEQ ID NO: 19) that is about 96% identical (98% similar) to mouse ptc (SEQ ID NO:09). The human patched gene (SEQ ID NO:18), including coding and non-coding sequences, is about 89% identical to the mouse patched gene (SEQ ID NO:09).

[0029] The butterfly PTC homolog (SEQ ID NO:4) is 1,300 amino acids long and overall has a 50% amino acid identity (72% similarity) to fly PTC (SEQ ID NO:6). With the exception of a divergent C-terminus, this homology is evenly spread across the coding sequence. A 267 bp exon from the beetle patched gene encodes an 89 amino acid protein fragment which was found to be 44% and 51% identical to the corresponding regions of fly and butterfly PTC respectively.

[0030] The mouse ptc message is about 8 kb long and the message is present in low levels as early as 7 dpc, the abundancy increasing by 11 and 15 dpc. Northern blot indicates a clear decrease in the amount of message at 17 dpc. In the adult, PTC RNA is present in high amounts in the brain and lung, as well as in moderate amounts in the kidney and liver. Weak signals are detected in heart, spleen, skeletal muscle and testes.

[0031] In mouse embryos, ptc mRNA is present at 7 dpc, using in situ hybridization. ptc is present at high levels along the neural axis of 8.5 dpc embryos. By 11.5 dpc, ptc can be detected in developing lung buds and gut, consistent with its Northern profile. In addition, the gene is present at high levels in the ventricular zone of the central nervous system as well as in the zona limitans of the prosencephalon. ptc is also strongly transcribed in the perichondrium condensing cartilage of 11.5 and 13.5 dpc limb buds, as well as in the ventral portion of the somites, a region which is prospective sclerotome and eventually forms bone in the vertebral column. PTC is present in a wide range of tissues from endodermal, mesodermal, as well as ectodermal origin, evidencing the fundamental role in many aspects of embryonic development, including the condensation of cartilage, the patterning of limbs, the differentiation of lung tissue, and the generation of neurons.

[0032] The patched nucleic acid may be used for isolating the gene from various mammalian sources of interest, particularly primate, more particularly human, or from domestic animals, both pet and farm, e.g. lagomorpha, rodentiae, porcine, bovine, feline, canine, ovine, equine, etc. By using probes, particularly labeled probes of DNA sequences, of the patched gene, one may be able to isolate mRNA or genomic DNA, which may be then used for identifying mutations, particularly associated with genetic diseases, such as spina bifida, limb defects, lung defects, problems with tooth development, liver and kidney development, peripheral nervous system development, and other sites where a patched gene is involved in regulation. The subject probes can also be used for identifying the level of expression in cells associated with the testis to determine the relationship with the level of expression and sperm production.

[0033] The gene or fragments thereof may be used as probes for identifying the 5′ non-coding region comprising the transcriptional initiation region, particularly the enhancer regulating the transcription of patched. By probing a genomic library, particularly with a probe comprising the 5′ coding region, one can obtain fragments comprising the 5′ non-coding region. If necessary, one may walk the fragment to obtain further 5′ sequence to ensure that one has at least a functional portion of the enhancer. It is found that the enhancer is proximal to the 5′ coding region, a portion being in the transcribed sequence and downstream from the promoter sequences. The transcriptional initiation region may be used for many purposes, studying embryonic development, providing for regulated expression of patched protein or other protein of interest during embryonic development or thereafter, and in gene therapy.

[0034] The gene may also be used for gene therapy, by transfection of the normal gene into embryonic stem cells or into mature cells. A wide variety of viral vectors can be employed for transfection and stable integration of the gene into the genome of the cells. Alternatively, micro-injection may be employed, fusion, or the like for introduction of genes into a suitable host cell. See, for example, Dhawan et al., Science 254, 1509-1512 (1991) and Smith et al., Molecular and Cellular Biology (1990) 3268-3271.

[0035] By providing for the production of large amounts of PTC protein, one can use the protein for identifying ligands which bind to the PTC protein. Particularly, one may produce the protein in cells and employ the polysomes in columns for isolating ligands for the PTC protein. One may incorporate the PTC protein into liposomes by combining the protein with appropriate lipid surfactants, e.g. phospholipids, cholesterol, etc., and sonicate the mixture of the PTC protein and the surfactants in an aqueous medium. With one or more established ligands, e.g. hedgehog, one may use the PTC protein to screen for antagonists which inhibit the binding of the ligand. In this way, drugs may be identified which can prevent the transduction of signals by the PTC protein in normal or abnormal cells.

[0036] The PTC protein, particularly binding fragments thereof, the gene encoding the protein, or fragments thereof, particularly fragments of at least about 18 nucleotides, frequently of at least about 30 nucleotides and up to the entire gene, more particularly sequences associated with the hydrophilic loops, may be employed in a wide variety of assays. In these situations, the particular molecules will normally be joined to another molecule, serving as a label, where the label can directly or indirectly provide a detectable signal. Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g. magnetic particles, and the like. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures. The assays may be used for detecting the presence of molecules which bind to the patched gene or PTC protein, in isolating molecules which bind to the patched gene, for measuring the amount of patched, either as the protein or the message, for identifying molecules which may serve as agonists or antagonists, or the like.

[0037] Various formats may be used in the assays. For example, mammalian or invertebrate cells may be designed where the cells respond when an agonist binds to PTC in the membrane of the cell. An expression cassette may be introduced into the cell, where the transcriptional initiation region of patched is joined to a marker gene, such as β-galactosidase, for which a substrate forming a blue dye is available. A 1.5 kb fragment that responds to PTC signaling has been identified and shown to regulate expression of a heterologous gene during embryonic development. When an agonist binds to the PTC protein, the cell will turn blue. By employing a competition between an agonist and a compound of interest, absence of blue color formation will indicate the presence of an antagonist. These assays are well known in the literature. Instead of cells, one may use the protein in a membrane environment and determine binding affinities of compounds. The PTC may be bound to a surface and a labeled ligand for PTC employed. A number of labels have been indicated previously. The candidate compound is added with the labeled ligand in an appropriate buffered medium to the surface bound PTC. After an incubation to ensure that binding has occurred, the surface may be washed free of any non-specifically bound components of the assay medium, particularly any non-specifically bound labeled ligand, and any label bound to the surface determined. Where the label is an enzyme, substrate producing a detectable product may be used. The label may be detected and measured. By using standards, the binding affinity of the candidate compound may be determined.

[0038] The availability of the gene and the protein allows for investigation of the development of the fetus and the role patched and other molecules play in such development. By employing antisense sequences of the patched gene, where the sequences may be introduced in cells in culture, or a vector providing for transcription of the antisense of the patched gene introduced into the cells, one can investigate the role the PTC protein plays in the cellular development. By providing for the PTC protein or fragment thereof in a soluble form which can compete with the normal cellular PTC protein for ligand, one can inhibit the binding of ligands to the cellular PTC protein to see the effect of variation in concentration of ligands for the PTC protein on the cellular development of the host. Antibodies against PTC can also be used to block function, since PTC is exposed on the cell surface.

[0039] The subject gene may also be used for preparing transgenic laboratory animals, which may serve to investigate embryonic development and the role the PTC protein plays in such development. By providing for variation in the expression of the PTC protein, employing different transcriptional initiation regions which may be constitutive or inducible, one can determine the developmental effect of the differences in PTC protein levels. Alternatively, one can use the DNA to knock out the PTC protein in embryonic stem cells, so as to produce hosts with only a single functional patched gene or where the host lacks a functional patched gene. By employing homologous recombination, one can introduce a patched gene, which is differentially regulated, for example, is expressed to the development of the fetus, but not in the adult. One may also provide for expression of the patched gene in cells or tissues where it is not normally expressed or at abnormal times of development. One may provide for mis-expression or failure of expression in certain tissue to mimic a human disease. Thus, mouse models of spina bifida or abnormal motor neuron differentiation in the developing spinal cord are made available. In addition, by providing expression of PTC protein in cells in which it is otherwise not normally produced, one can induce changes in cell behavior upon binding of ligand to the PTC protein.

[0040] Areas of investigation may include the development of cancer treatments. The wingless gene, whose transcription is regulated in flies by PTC, is closely related to a mammalian oncogene, Wnt-1, a key factor in many cases of mouse breast cancer. Other Wnt family members, which are secreted signaling proteins, are implicated in many aspects of development. In flies, the signaling factor decapentaplegic, a member of the TGF-beta family of signaling proteins, known to affect growth and development in mammals, is also controlled by PTC. Since members of both the TGF-beta and Wnt families are expressed in mice in places close to overlapping with patched, the common regulation provides an opportunity in treating cancer. Also, for repair and regeneration, proliferation competent cells making PTC protein can find use to promote regeneration and healing for damaged tissue, which tissue may be regenerated by transfecting cells of damaged tissue with the ptc gene and its normal transcription initiation region or a modified transcription initiation region. For example, PTC may be useful to stimulate growth of new teeth by engineering cells of the gums or other tissues where PTC protein was during an earlier developmental stage or is expressed.

[0041] Since Northern blot analysis indicates that ptc is present at high levels in adult lung tissue, the regulation of ptc expression or binding to its natural ligand may serve to inhibit proliferation of cancerous lung cells. The availability of the gene encoding PTC and the expression of the gene allows for the development of agonists and antagonists. In addition, PTC is central to the ability of neurons to differentiate early in development. The availability of the gene allows for the introduction of PTC into host diseased tissue, stimulating the fetal program of division and/or differentiation. This could be done in conjunction with other genes which provide for the ligands which regulate PTC activity or by providing for agonists other than the natural ligand.

[0042] The availability of the coding region for various ptc genes from various species, allows for the isolation of the 5′ non-coding region comprising the promoter and enhancer associated with the ptc genes, so as to provide transcriptional and post-transcriptional regulation of the ptc gene or other genes, which allow for regulation of genes in relation to the regulation of the ptc gene. Since the ptc gene is autoregulated, activation of the ptc gene will result in activation of transcription of a gene under the transcriptional control of the transcriptional initiation region of the ptc gene. The transcriptional initiation region may be obtained from any host species and introduced into a heterologous host species, where such initiation region is functional to the desired degree in the foreign host. For example, a fragment of from about 1.5 kb upstream from the initiation codon, up to about 10 kb, preferably up to about 5 kb may be used to provide for transcriptional initiation regulated by the PTC protein, particularly the Drosophila 5′-non-coding region (GenBank accession no. M28418).

[0043] The following examples are offered by illustration not by way of limitation.

EXPERIMENTAL

[0044] Methods and Materials

[0045] I. PCR on Mosquito (Anopheles gambiae) Genomic DNA:

[0046] PCR primers were based on amino acid stretches of fly PTC that were not likely to diverge over evolutionary time and were of low degeneracy. Two such primers (P2R1 (SEQ ID NO: 14): GGACGAATTCAARGTNCAYCARYTNTGG, P4R1: (SEQ ID NO: 15) GGACGAATTCCYTCCCARAARCANTC, (the underlined sequences are Eco RI linkers) amplified an appropriately sized band from mosquito genomic DNA using the PCR. The program conditions were as follows:

[0047] 94° C. 4 min.; 72° C. Add Taq;

[0048] [49° C. 30 sec.; 72° C. 90 sec.; 94° C. 15 sec] 3 times

[0049] [94° C. 15 sec.; 50° C. 30 sec.; 72° C. 90 sec] 35 times

[0050] 72° C. 10 min; 4° C. hold

[0051] This band was subcloned into the EcoRV site of pBluescript II and sequenced using the USB Sequence kit.

[0052] II. Screen of a Butterfly cDNA Library with Mosquito PCR Product

[0053] Using the mosquito PCR product (SEQ ID NO:7) as a probe, a 3 day embryonic Precis coenia λgt10 cDNA library (generously provided by Sean Carroll) was screened. Filters were hybridized at 65° C. overnight in a solution containing 5×SSC, 10% dextran sulfate, 5×Denhardt's, 200 μg/ml sonicated salmon sperm DNA, and 0.5% SDS. Filters were washed in 0.1×SSC, 0.1% SDS at room temperature several times to remove nonspecific hybridization. Of the 100,000 plaques initially screened, 2 overlapping clones, L1 and L2, were isolated, which corresponded to the N terminus of butterfly PTC. Using L2 as a probe, the library filters were rescreened and 3 additional clones (L5, L7, L8) were isolated which encompassed the remainder of the ptc coding sequence. The full length sequence of butterfly ptc (SEQ ID NO:3) was determined by ABI automated sequencing.

[0054] III. Screen of a Tribolium (beetle) Genomic Library with Mosquito PCR Product and 900 bp Fragment from the Butterfly Clone

[0055] A λgem11 genomic library from Tribolium casteneum (gift of Rob Dennell) was probed with a mixture of the mosquito PCR (SEQ ID NO:7) product and BstXI/EcoRI fragment of L2. Filters were hybridized at 55° C. overnight and washed as above. Of the 75,000 plaques screened, 14 clones were identified and the SacI fragment of T8 (SEQ ID NO: 1), which crosshybridized with the mosquito and butterfly probes, was subcloned into pBluescript.

[0056] IV. PCR on Mouse cDNA Using Degenerate Primers Derived from Regions Conserved in the Four Insect Homologues

[0057] Two degenerate PCR primers (P4REV: (SEQ ID NO:16) GGACGAATTCYTNGANTGYTTYTGGGA; P22: (SEQ ID NO: 17) CATACCAGCCAAGCTTGTCIGGCCARTGCAT) were designed based on a comparison of PTC amino acid sequences from fly (Drosophila melanogaster) (SEQ ID NO:6), mosquito (Anopheles gambiae)(SEQ ID NO:8), butterfly (Precis coenia)(SEQ ID NO:4), and beetle (Tribolium casteneum)(SEQ ID NO:2). I represents inosine, which can form base pairs with all four nucleotides. P22 was used to reverse transcribe RNA from 12.5 dpc mouse limb bud (gift from David Kingsley) for 90 min at 37° C. PCR using P4REV(SEQ ID NO:17) and P22(SEQ ID NO: 18) was then performed on 1 μl of the resultant cDNA under the following conditions:

[0058] 94° C. 4 min.; 72° C. Add Taq;

[0059] [94° C. 15 sec.; 50° C. 30 sec.; 72° C. 90 sec.] 35 times

[0060] 72° C. 10 min.; 4° C. hold

[0061] PCR products of the expected size were subcloned into the TA vector (Invitrogen) and sequenced with the Sequenase Version 2.0 DNA Sequencing Kit (U.S.B.).

[0062] Using the cloned mouse PCR fragment as a probe, 300,000 plaques of a mouse 8.5 dpc λgt10 cDNA library (a gift from Brigid Hogan) were screened at 65° C. as above and washed in 2×SSC, 0.1% SDS at room temperature. 7 clones were isolated, and three (M2 M4, and M8) were subcloned into pBluescript II. 200,000 plaques of this library were rescreened using first, a 1.1 kb EcoRI fragment from M2 to identify 6 clones (M9-M16) and secondly a mixed probe containing the most N terminal (XhoI fragment from M2) and most C terminal sequences (BamHI/BglII fragment from M9) to isolate 5 clones (M17-M21). M9, M10, M14, and M17-21 were subcloned into the EcoRI site of pBluescript II (Strategene).

[0063] V. RNA Blots and in situ Hybridizations in Whole and Sectioned Mouse Embryos Northerns:

[0064] A mouse embryonic Northern blot and an adult multiple tissue Northern blot (obtained from Clontech) were probed with a 900 bp EcoRI fragment from an N terminal coding region of mouse ptc. Hybridization was performed at 65° C. in 5×SSPE, 10×Denhardt's, 100 μg/ml sonicated salmon sperm DNA, and 2% SDS. After several short room temperature washes in 2×SSC, 0.05% SDS, the blots were washed at high stringency in 0.1×SSC, 0.1% SDS at 50C.

[0065] In situ hybridization of sections:

[0066] 7.75, 8.5, 11.5, and 13.5 dpc mouse embryos were dissected in PBS and frozen in Tissue-Tek medium at −80° C. 12-16 μm frozen sections were cut, collected onto VectaBond (Vector Laboratories) coated slides, and dried for 30-60 minutes at room temperature. After a 10 minute fixation in 4% paraformaldehyde in PBS, the slides were washed 3 times for 3 minutes in PBS, acetylated for 10 minutes in 0.25% acetic anhydride in triethanolamine, and washed three more times for 5 minutes in PBS. Prehybridization (50% formamide, 5×SSC, 250 μg/ml yeast tRNA, 500 μg/ml sonicated salmon sperm DNA, and 5×Denhardt's) was carried out for 6 hours at room temperature in 50% formamide/5×SSC humidified chambers. The probe, which consisted of 1 kb from the N-terminus of ptc, was added at a concentration of 200-1000 ng/ml into the same solution used for prehybridization, and then denatured for five minutes at 80° C. Approximately 75 μl of probe were added to each slide and covered with Parafilm. The slides were incubated overnight at 65° C. in the same humidified chamber used previously. The following day, the probe was washed successively in 5×SSC (5 minutes, 65° C.), 0.2×SSC (1 hour, 65° C.), and 0.2×SSC (10 minutes, room temperature). After five minutes in buffer BI (0.1M maleic acid, 0.15 M NaCl, pH 7.5), the slides were blocked for 1 hour at room temperature in 1% blocking reagent (Boerhinger-Mannheim) in buffer B1, and then incubated for 4 hours in buffer B1 containing the DIG-AP conjugated antibody (Boerhinger-Mannheim) at a 1:5000 dilution. Excess antibody was removed during two 15 minute washes in buffer B 1, followed by five minutes in buffer B3 (100 mM Tris, 100 mM NaCl, 5 mM MgCl₂, pH 9.5). The antibody was detected by adding an alkaline phosphatase substrate (350 μl 75 mg/ml X-phosphate in DMF, 450 μl 50 mg/ml NBT in 70% DMF in 100 mls of buffer B3) and allowing the reaction to proceed over-night in the dark. After a brief rinse in 10 mM Tris, 1 mM EDTA, pH 8.0, the slides were mounted with Aquamount (Lerner Laboratories).

[0067] VI. Drosophila 5-Transcriptional Initiation Region β-gal Constructs.

[0068] A series of constructs were designed that link different regions of the ptc promoter from Drosophila to a LacZ reporter gene in order to study the cis regulation of the ptc expression pattern. See FIG. 1. A 10.8 kb BamHI/BspM1 fragment comprising the 5′-non-coding region of the mRNA at its 3′-terminus was obtained and truncated by restriction enzyme digestion as shown in FIG. 1. These expression cassettes were introduced into Drosophila lines using a P-element vector (Thummel et al., Gene 74, 445-456 (1988), which were injected into embryos, providing flies which could be grown to produce embryos. (See Spradling and Rubin, Science (1982) 218, 341-347 for a description of the procedure.) The vector used a pUC8 background into which was introduced the white gene to provide for yellow eyes, portions of the P-element for integrtion, and the constructs were inserted into a polylinker upstream from the LacZ gene. The resulting embryos were stained using antibodies to LacZ protein conjugated to HRP and the embryos developed with OPD dye to identify the expression of the LacZ gene. The staining pattern is described in FIG. 1, indicating whether there was staining during the early and late development of the embryo.

[0069] VII. Isolation of a Mouse ptc Gene

[0070] Homologues of fly PTC (SEQ ID NO:6) were isolated from three insects: mosquito, butterfly and beetle, using either PCR or low stringency library screens. PCR primers to six amino acid stretches of PTC of low mutatability and degeneracy were designed. One primer pair, P2 and P4, amplified an homologous fragment of ptc from mosquito genomic DNA that corresponded to the first hydrophilic loop of the protein. The 345 bp PCR product (SEQ ID NO:7) was subcloned and sequenced and when aligned to fly PTC, showed 67% amino acid identity.

[0071] The cloned mosquito fragment was used to screen a butterfly λGT 10 cDNA library. Of 100,000 plaques screened, five overlapping clones were isolated and used to obtain the full length coding sequence. The butterfly PTC homologue (SEQ ID NO:4) is 1,311 amino acids long and overall has 50% amino acid identity (72% similarity) to fly PTC. With the exception of a divergent C-terminus, this homology is evenly spread across the coding sequence. The mosquito PCR clone (SEQ ID NO:7) and a corresponding fragment of butterfly cDNA were used to screen a beetle λgem11 genomic library. Of the plaques screened, 14 clones were identified. A fragment of one clone (T8), which hybridized with the original probes, was subcloned and sequenced. This 3 kb piece contains an 89 amino acid exon (SEQ ID NO:2) which is 44% and 51% identical to the corresponding regions of fly and butterfly PTC respectively.

[0072] Using an alignment of the four insect homologues in the first hydrophilic loop of the PTC, two PCR primers were designed to a five and six amino acid stretch which were identical and of low degeneracy. These primers were used to isolate the mouse homologue using RT-PCR on embryonic limb bud RNA. An appropriately sized band was amplified and upon cloning and sequencing, it was found to encode a protein 65% identical to fly PTC. Using the cloned PCR product and subsequently, fragments of mouse ptc cDNA, a mouse embryonic λcDNA library was screened. From about 300,000 plaques, 17 clones were identified and of these, 7 form overlapping cDNA's which comprise most of the protein-coding sequence (SEQ ID NO:9).

[0073] VIIa. Developmental and Tissue Distribution of Mouse PTC RNA

[0074] In both the embryonic and adult Northern blots, the ptc probe detects a single 8 kb message. Further exposure does not reveal any additional minor bands. Developmentally, ptc mRNA is present in low levels as early as 7 dpc and becomes quite abundant by 11 and 15 dpc. While the gene is still present at 17 dpc, the Northern blot indicates a clear decrease in the amount of message at this stage. In the adult, ptc RNA is present in high amounts in the brain and lung, as well as in moderate amounts in the kidney and liver. Weak signals are detected in heart, spleen, skeletal muscle, and testes.

[0075] VIIb. In situ Hybridization of Mouse PTC in Whole and Section Embryos

[0076] Northern analysis indicates that ptc mRNA is present at 7 dpc, while there is no detectable signal in sections from 7.75 dpc embryos. This discrepancy is explained by the low level of transcription. In contrast, ptc is present at high levels along the neural axis of 8.5 dpc embryos. By 11.5 dpc, ptc can be detected in the developing lung buds and gut, consistent with its adult Northern profile. In addition, the gene is present at high levels in the ventricular zone of the central nervous system, as well as in the zona limitans of the prosencephalon. ptc is also strongly transcribed in the condensing cartilage of 11.5 and 13.5 dpc limb buds, as well as in the ventral portion of the somites, a region which is prospective sclerotome and eventually forms bone in the vertebral column. ptc is present in a wide range of tissues from endodermal, mesodermal and ectodermal origin supporting its fundamental role in embryonic development.

[0077] VIII. Isolation of the Human ptc Gene

[0078] To isolate human ptc (hptc), 2×10⁵ plaques from a human lung cDNA library (HL3022a, Clonetech) were screened with a 1 kbp mouse ptc fragment, M2-2. Filters were hybridized overnight at reduced stringency (60° C. in 5×SSC, 10% dextran sulfate, 5×Denhardt's, 0.2 mg/ml sonicated salmon sperm DNA, and 0.5% SDS). Two positive plaques (H1 and H2) were isolated, the inserts cloned into pBluescript, and upon sequencing, both contained sequence highly similar to the mouse ptc homolog. To isolate the 5′ end, an additional 6×10⁵ plaques were screened in duplicate with M2-3 EcoR I and M2-3 Xho I (containing 5′ untranslated sequence of mouse ptc) probes. Ten plaques were purified and of these, 6 inserts were subloned into pBluescript. To obtain the full coding sequence, H2 was fully and H14, H20, and H21 were partially sequenced. The 5.1 kbp of human ptc sequence (SEQ ID NO: 18) contains an open reading frame of 1447 amino acids (SEQ ID NO: 19) that is 96% identical and 98% similar to mouse ptc. The 5′ and 3′ untranslated sequences of human ptc (SEQ ID NO: 18) are also highly similar to mouse ptc (SEQ ID NO:09) suggesting conserved regulatory sequence.

[0079] IX. Comparison of Mouse, Human, Fly and Butterfly Sequences

[0080] The deduced mouse PTC protein sequence (SEQ ID NO: 10) has about 38% identical amino acids to fly PTC over about 1,200 amino acids. This amount of conservation is dispersed through much of the protein excepting the C-terminal region. The mouse protein also has a 50 amino acid insert relative to the fly protein. Based on the sequence conservation of PTC and the functional conservation of hedgehog between fly and mouse, one concludes that ptc functions similarly in the two organisms. A comparison of the amino acid sequences of mouse (mptc) (SEQ ID NO: 10), human (hptc) (SEQ ID NO: 19), butterfly (bptc)(SEQ ID NO:4) and drosophila (ptc) (SEQ ID NO:6) is shown in Table 1. TABLE 1 alignment of human, mouse, fly, and butterfly PTC homologs alignment of human, mouse, fly, and butterfly ptc homologs HPTC MASAGNAAEPQDR--GGGGSGCIGAPGRPAGGGRRRRTGGLRRAAAPDRDYLHRPSYCDA MPTC MASAGNAA-------------GALGRQAGGGRRRRTGGPHRA-APDRDYLHRPSYCDA PTC M-----DRDSLPRVPDTHGD--VVDE---------KLFSDL---------YI-RTSWVDA BPTC MVAPDSEAPSNPRITAAHESPCATEA---------RHSADL---------YI-RTSWVDA *                                  .  ..          *. * *  ** HPTC AFALEQISKGKTGRKAPLWLRAKFQRLLFKLGCYIQKNCGKFLVVGLLIFGAFAVGLKA MPTC AFALEQISKGKATGRKAPLWLRAKFQRLLFKLGCYIQKNCGKFLVVGLLIFGAFAVGLKA PTC QVALDQIDKGKARGSRTAIYLRSVFQSHLETLGSSVQKHAGKVLFVAILVLSTFCVGLKS BPTC ALALSELEKGNIEGGRTSLWIRAWLQEQLFILGCFLQGDAGKVLFVAILVLSTFCVGLKS   * .. **.  * .... .*. *  *  **  .* . ** * *..*....* ****. HPTC ANLETNVEELWVEVGGRVSRELNYTRQKIGEEAMFNPQLMIQTPKEEGANVLTTEALLQH MPTC ANLETNVEELWVEVGGRVSRELNYTRQKIGEEAMFNPQLMIQTPKEEGANVLTTEALLQH PTC AQIHSKVHQLWIQEGGRLEAELAYTQKTIGEDESATHQLLIQTTHDPNASVLHPQALLAH BPTC AQIHTRVDQLWVQEGGRLEAELKYTAQALGEADSSTHQLVIQTAKDPDVSLLHPGALLEH *.... *..**.. ***. ** ** . .**      **.***  .   ..*   *** * HPTC LDSALQASRVHVYMYNRQWKLEHLCYKSGELITET-GYMDQIIEYLYPCLIITPLDCFWE MPTC LDSALQASRVHVYMYNRQWKLEHLCYKSGELITET-GYMDQIIEYLYPCLIITPLDCFWE PTC LEVLVKATAVKVHLYDTEWGLRDMCNMPSTPSFEGIYYIEQILRHLIPCSIITPLDCFWE BPTC LKVVHAATRVTVHMYDIEWRLKDLCYSPSIPDFEGYHHIESIIDNVIPCAIITPLDCFWE *     *. * * .*. .* * ..*  ..    *    .. *.  . ** ********** HPTC GAKLQSGTAYLLGKPPLR----WTNFDPLEFLEELK------KINYQVDSWEEMLNKAEV MPTC GAKLQSGTAYLLGKPPLR----WTNFDPLEFLEELK------KINYQVDSWEEMLNKAEV PTC GSQLL-GPESAVVIPGLNQRLLWTTLNPASVMQYMKQKMSEEKISFDFETVEQYNKRAAI BPTC GSKLL-GPDYPIYVPHLKHKLQWTHLNPLEVVEEVK-KL---KFQFPLSTIEAYMKRAGI *..*  *    .  * *     ** ..*   .. .*      *. .   . *  ...* . HPTC GHGYMDRPCLNPADPDCPATAPNKNSTKPLDMALVLNGGCHGLSRKYMHWQEELIVGGTV MPTC GHGYMRPCLNPADPDCPATAPNKNSTKPLDVALVLNGGCQGLSRKYMHWQEELIVGGTV PTC GSGYMEKPCLNPLNPNCPDTAPNKNSTQPPDVGAILSGGCYGYAAKHMRWPEELIVGGRK BPTC TSAYMKKPCLDPTDPHCPATAPNKKSGHIPDVAAELSHGCYGFAAAYMHHWPEQLIVGGAT   .** .***.* .*.** *****.*    *..  *..  *. ** * .   *** * ***** HPTC KNSTGKLVSAHALQTMFQLMTPKQMYEHFKGYEYVSHINWNEDKAAAILEAWQRTYVEVV MPTC KNATGKLVSAHALQTMFQLMTPKQMYEHFRGYDYVSHINWNEDRAAAILEAWQRTYVEVV PTC RNRSGHLRKAQALQSVVQLMTEKEMYDQWQDNYKVHHLGWTQEKAAEVLNAWQRNFSREV BPTC RNSTSARSARALQTVQLMGEREMYEYWADHYKVHQIGWNQEKAAAVLDAWQRKFAAEV .* .. *  *.***.. ***  ..**.   .   * .. * ...** .*.**** .   * HPTC HQSVAQNSTQK----VLSFTTTTLDDILKSFSDVSVIRVASGYLLMLAYACLTMLRW-DC MPTC HQSVAPNSTQK----VLPFTTTTLDDILKSFSDVSVIRVASGYLLMLAYACLTMLRW-DC PTC EQLLRKQSRIATNYDIYVFSSAALDDILAKFSHPSALSIVIGVAVTVLYAFCTLLRWRDP BPTC RKI-TTSGSVSSAYSFYPFSTSTLNDILGKFSEVSLKNIILGYMFMLIYVAVTLIQWRDP .     .          *....*.***  **. *   .  *    . *  *...* * HPTC SKSQGAVGLAAGVLLVALSVAGLGLCSLIGISFMAATTQVLPFLALGVGVDDVFLLAHAF MPTC SKSQGAVGLAGVLLVALSVAAGLGLCSLIGISFNAATTQVLPFLALGVGVDDVFLLAHAF PTC VRGQSSVGVAGVLLMCFSTAAGLGLSALLGIVFNAASTQVVPFLALGLGVDHIFMLTAAY BPTC IRSQAGVGIAGVLLLSITVAAGLGFCALLGIPFNASSTQIVPFLALGLGVQDMFLLTHTY ..*..**.*****. .. *****. .*.** ***..**..******.**...*.*. .. HPTC SETGQNKRIPFEDRTGECLKRTGASVALTSISNVTAFFMAALIPIPALRAFSLQAAVVVV MPTC SETGQNKRIPFEDRTGECLKRTGASVALTSISNVTAFFMAALIPIPALRAFSLQAAVWV PTC AESN------RREQTKLILKKVGPSILFSACSTAGSFFAAAFIPVPALKVFCLQAAIVMC BPTC VEQAGD--VPREERTGLVLKKSGLSVLLASLCNVMAFLAAALLPIPAFRVFCLQAAILLL *          ..*   **. * *. ...     .*. **..*.**.. * ****... HPTC FNFAMVLLIFPAILSMDLYRREDRRLDIFCCFTSPCVSRVIQVEPQAYTDTHDNTRYSPP MPTC FNFAMVLLIFPAILSMDLYRREDRRLDIFCCFTSFCVSRVIQVEPQAYTEPHSNTRYSPP PTC SNLAAALLVFPAMISLDLRRRTAGRADIFCCCF-PVWKEQPKVAPPVLPLNNNNGR--- BPTC FNLGSILLVFPAMISLDLRRRSAARADLLCCLM-P---ESP------LPKKKI-PER---- *..  **.***..*.** **   * *..**   *                    * HPTC PPYSSHSFAHETQITMQSTVQLRTEYDPHVYYTTAEPRSEISVQPVTVTQDT LSCQSP MPTC PPYTSHSFAHETHITMQSTVQLRTEYDPHTHVYYTTAEPRSEISVQPVTVTQDNLSCQSP PTC ----------------------------------GARHPKSCNNNRVPLPAQNPLLEQRA BPTC -----------------------------------AKTRKNDKTHRID-TTRQPLDPDVS                                    .   ..    .    ... *  . . HPTC ESTSSTRDLLSQFSDSSLHCLEFPCTKWTLSSFAEKHYAPFLLKPKAKVVVIFLFLGLLG MPTC ESTSSTRDLLSQFSDSSLHCLEPPCTKWTLSSFAEKHYAPFLLKPKAKVVVILLFLGLLG PTC DIPGSS------------HSLASF----SLATFAFQHYTPFLMRSWVKFLTVMGFLAALI BPTC ENVTKT------------CCL-SV----SLTKWAKNQYAPFIMRPAVKVTSMLALIAVIL .    .              * .     .*.  * ..*.**....  *   .  ... . HPTC VSLYGTTRVRDGLDLTDIVPRETREYDFIAAQFKYFSFYNMYIVTQKA-DYPNIQHLLYD MPTC VSLYGTTRVRDGLDLTDIVPRETREYDFIAAQFKYFSFYNMYIVTQKA-DYPNIQHLLYD PTC SSLYASTRLQDGLDIIDLVPKDSNEHKFLDAQTRLFGFYSMYAVTQGNFEYPTQQQLLRD BPTC TSVWGATKVKDGLDLTDIVPENTDEHEFLSRQEKYFGFYNMYAVTQGNFEYPTNQKLLYE *. ..*...****. *.** .. *  *.  * . *.**.** ***   .**  * ** . HPTC LHRSFSNVKYVMLEENKQLPKMWLHYFRDWLQGLQDAFDSDWETGKIMPNN-YKNGSDDG MPTC LHKSFSNVKYVMLEENKQLPQMWLHYFRDWLQGLQDAFDSDWZTGRIMPNN-YKNGSDDG PTC YHDSFVRVPHVIKNDNGGLPDFWLLLFSEWLGNLQKIFDEEYRDGRLTKECWFPNASSDA BPTC YHDQFVRIPNIIKNDNGGLTKFWLSLFRDWLLDLQVAFDKEVASGCITQEYWCKNASDEG *  *  .  .. ..*  *   **  *  **  **  ** .   * .  .    *.* .. HPTC VLAYKLLVQTGSRDKPIDISQLTK-QRLVDADGIINPSAFYIYLTAWVSNDPVAYAASQA MPTC VLAYKLLVQTGSRDKPIDISQLTK-QRLVDADGIINPSAFYIYLTAWVSNDPVAYAASQA PTC ILAYKLIVQTGHVDNPVDKELVLT-NRLVNSDGIINQRAFYNYLSAWATNDVEAYGASQG BPTC ILAYKLMVQTGHVDNPIDKSLITAGHRLVDKDGIINPKAFYNYLSAWATNDALAYGASQG *****.****  *.*.*   .   .***. *****  ***  **.** .**  **.*** HPTC NIRPHRPEWVHDKADYMPETRLRIPAAEPIEYAQFPFYLNGLRDTSDFVEAIEKVRTICS MPTC NIRPHRPEWVHDKADYMPETRLRIPAAEPIEYAQFPFYLNGLRDTSDFVEAIEKVRVICN PTC KLYPEPRQYFHQPNEY----DLKIPKSLPLVYAQMPFYLHGLTDTSQIKTLIGEIRDLSV BPTC NLKPQPQRWIHSPEDV----HLEIKKSSPLIYTQLPFYLSGLSDTDSIKTLIRSVRDLCL .. *.     *   .//////* *  . *. *.* **** ** **  .   *  .* . HPTC NYTSLGLSSYPNGYPFLFWEQYIGLRHWLLLFISVVLACTFLVCAVFLLNPWTAGIIVMV MPTC NYTSLGLSSYPNGYPFLFWEQYISLRHWLLLSISVVLACTFLVCAVFLLNPWTAGIIVMV PTC KYEGFGLPNYPSGIPFIFWEQYMTLRSSLAMILACVLLAALVLVSLLLLSVWAAVLVILS BPTC KYEAKGLPNFPSGIPFLFWEQYLYLRTSLLLALACALGAVFIAVMVLLLNAWAAVLVTLA .* . **...*.* **.*****. **  * . ..  *   ..   ..**. *.* .. . HPTC LALMVTELFGMMGLIGIKLSAVPVVILIASVGIGVEFTVHVALAFLTAIGDKNRRAVLAL MPTC LALMTVELFGMMGLIGIKLSAVPVVILIASVGIGVEFTVHVALAFLTAIGDKNHRAMLAL PTC VLASLAQIFGAMTLLGIKLSAIPAVILILSVGMMLCFNVLISLGFMTSVGNRQRRVQLSM BPTC LATLVLQLLGVMALLGVKLSAMPPVLLVLAIGRGVHFTVHLCLGFVTSIGCKRRRASLAL .     ...* * *.*.****.* ***. ..*  . * * . *.*.*..* . .*  *.. HPTC EHMFAPVLDGAVSTLLGVLMLAGSEFDFIVRYFFAVLAILTILGVLNGLVLLPVLLSFFG MPTC EHMFAPVLDGAVSTLLGVLMLAGSEFDFIVRYFFAVLAILTVLGVLNGLVLLPVLLSFFG PTC QMSLGPLVHGMLTSGVAVFMLSTSPFEFVIRHFCWLLLVVLCVGACNSLLVFPILLSMVG BPTC ESVLAPVVHGALAALAASMLAASEFGFVARLFLRLLLALVFLGLIDGLLFFPIVLSILG .  ..*...* ... ..  **. * * *. * *  .*  .  .*  ..*. .*..**  * HPTC PYPEVSPANGLNRLPTPSPEPPPSVVRFAMPPGHTHSGSDSSDSEYSSQTTVSGLSE-EL MPTC PCPEVSPANGLNRLPTPSPEPPPSVYRFAVPPGHTNNGSDSSDSEYSSQTTVSGISE-EL PTC PEAELVPLEHPDRISTPSFLPVRSSKRSGKSYVVQGSRSSRGSCQKSNHHHHKDLNDPSL BPTC PAAEVRPIEHPERLSTPSPKCSPIHPRKSSSSSGGGDKSSRTS--KSAPRPC----APSL * .*. * .  .*..****       * . .       *      *            * HPTC RHYEAQQGAGGPAHQVIVEATENPVFAHSTVVHPESRHHPPSNPRQQPHLDSGSLPPGRQ MPTC RQYEAQQGAGGPAHQVIVEATENPVFARSTVVHPDSRHQPPLTPRQQPHLDSGSLSPGRQ PTC TTITEEPQSWKSSNSSIQMFNDWTYQFREQ--RPASYAAPPPAYHKAAAQQHHQEQGPPT BPTC TTITEEPSSWHSSAHSVQSSMQSIVVQFEVVVETTTYNGSDSASGRSTPTKSSHGGAITT      .  .  ..   .  . .            .   .     . HPTC GQQPRRDPPREGLWPPLYRPRRDAFEISTEGHSGPSNRARWGPRGARSHNPRNPASTAMG MPTC GQQPRRDPPREGLRPPPYRPRRDAFEISTEGHSGPSNRDRSGPRGARSHNPRNPTSTAMG PTC TPPPPFPTA-----------------YPPELQSIVVQPEVTVETTHS-----------DS BPTC TKVTATANIKVEVVTPSDRKSRRSYHYYDRRRDRDEDRDRDRERDRDRDRDRDRDRDRDR                                .      . HPTC SSVPGYCQPITTVTASASVTVAVHPPPVPGPGRNPRGGLCPGY---PETDHGLEEDPHVP MPTC SSVPSYCQPITTVTASASVTVAVHPP--PGPGRNPRGGFCFGYESYPETDHGVFEDPHVP PTC NT--------TKVTATANIKVELAMP-----GRAVRS---YNFTS--------------- BPTC DR--------DRERSRERDRRDRYRD-----ERDHRA---SPRENGRDSGHE--------               .                 *  *. HPTC FHVRCERRDSKVEVIELQDVECEERPRGSSSN MPTC FHVRCERRDSKVEVIELQDVECEERPWGSSSN PTC ------------------------------- BFTC --------------------------SDSSRH

[0081] The identity of ten other clones recovered from the mouse library is not determined. These cDNAs cross-hybridize with mouse ptc sequence, while differing as to their restriction maps. These genes encode a family of proteins related to the patched protein. Alignment of the human and mouse nucleotide sequences, which includes coding and noncoding sequence, reveals 89% identity.

[0082] In accordance with the subject invention, mammalian patched genes, including the mouse and human genes, are provided which allow for high level production of the patched protein, which can serve many purposes. The patched protein may be used in a screening for agonists and antagonists, for isolation of its ligand, particularly hedgehog, more particularly Sonic hedgehog, and for assaying for the transcription of the mRNA ptc. The protein or fragments thereof may be used to produce antibodies specific for the protein or specific epitopes of the protein. In addition, the gene may be employed for investigating embryonic development, by screening fetal tissue, preparing transgenic animals to serve as models, and the like.

[0083] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0084] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

1 19 736 base pairs nucleic acid single linear DNA (genomic) 1 AACNNCNNTN NATGGCACCC CCNCCCAACC TTTNNNCCNN NTAANCAAAA NNCCCCNTTT 60 NATACCCCCT NTAANANTTT TCCACCNNNC NNAAANNCCN CTGNANACNA NGNAAANCCN 120 TTTTTNAACC CCCCCCACCC GGAATTCCNA NTNNCCNCCC CCAAATTACA ACTCCAGNCC 180 AAAATTNANA NAATTGGTCC TAACCTAACC NATNGTTGTT ACGGTTTCCC CCCCCAAATA 240 CATGCACTGG CCCGAACACT TGATCGTTGC CGTTCCAATA AGAATAAATC TGGTCATATT 300 AAACAAGCCN AAAGCTTTAC AAACTGTTGT ACAATTAATG GGCGAACACG AACTGTTCGA 360 ATTCTGGTCT GGACATTACA AAGTGCACCA CATCGGATGG AACCAGGAGA AGGCCACAAC 420 CGTACTGAAC GCCTGGCAGA AGAAGTTCGC ACAGGTTGGT GGTTGGCGCA AGGAGTAGAG 480 TGAATGGTGG TAATTTTTGG TTGTTCCAGG AGGTGGATCG TCTGACGAAG AGCAAGAAGT 540 CGTCGAATTA CATCTTCGTG ACGTTCTCCA CCGCCAATTT GAACAAGATG TTGAAGGAGG 600 CGTCGAANAC GGACGTGGTG AAGCTGGGGG TGGTGCTGGG GGTGGCGGCG GTGTACGGGT 660 GGGTGGCCCA GTCGGGGCTG GCTGCCTTGG GAGTGCTGGT CTTNGCGNGC TNCNATTCGC 720 CCTATAGTNA GNCGTA 736 107 amino acids amino acid single linear protein 2 Xaa Pro Pro Pro Asn Tyr Asn Ser Xaa Pro Lys Xaa Xaa Xaa Leu Val 1 5 10 15 Leu Thr Pro Xaa Val Val Thr Val Ser Pro Pro Lys Tyr Met His Trp 20 25 30 Pro Glu His Leu Ile Val Ala Val Pro Ile Arg Ile Asn Leu Val Ile 35 40 45 Leu Asn Lys Pro Lys Ala Leu Gln Thr Val Val Gln Leu Met Gly Glu 50 55 60 His Glu Leu Phe Glu Phe Trp Ser Gly His Tyr Lys Val His His Ile 65 70 75 80 Gly Trp Asn Gln Glu Lys Ala Thr Thr Val Leu Asn Ala Trp Gln Lys 85 90 95 Lys Phe Ala Gln Val Gly Gly Trp Arg Lys Glu 100 105 5187 base pairs nucleic acid single linear cDNA 3 GGGTCTGTCA CCCGGAGCCG GAGTCCCCGG CGGCCAGCAG CGTCCTCGCG AGCCGAGCGC 60 CCAGGCGCGC CCGGAGCCCG CGGCGGCGGC GGCAACATGG CCTCGGCTGG TAACGCCGCC 120 GGGGCCCTGG GCAGGCAGGC CGGCGGCGGG AGGCGCAGAC GGACCGGGGG ACCGCACCGC 180 GCCGCGCCGG ACCGGGACTA TCTGCACCGG CCCAGCTACT GCGACGCCGC CTTCGCTCTG 240 GAGCAGATTT CCAAGGGGAA GGCTACTGGC CGGAAAGCGC CGCTGTGGCT GAGAGCGAAG 300 TTTCAGAGAC TCTTATTTAA ACTGGGTTGT TACATTCAAA AGAACTGCGG CAAGTTTTTG 360 GTTGTGGGTC TCCTCATATT TGGGGCCTTC GCTGTGGGAT TAAAGGCAGC TAATCTCGAG 420 ACCAACGTGG AGGAGCTGTG GGTGGAAGTT GGTGGACGAG TGAGTCGAGA ATTAAATTAT 480 ACCCGTCAGA AGATAGGAGA AGAGGCTATG TTTAATCCTC AACTCATGAT ACAGACTCCA 540 AAAGAAGAAG GCGCTAATGT TCTGACCACA GAGGCTCTCC TGCAACACCT GGACTCAGCA 600 CTCCAGGCCA GTCGTGTGCA CGTCTACATG TATAACAGGC AATGGAAGTT GGAACATTTG 660 TGCTACAAAT CAGGGGAACT TATCACGGAG ACAGGTTACA TGGATCAGAT AATAGAATAC 720 CTTTACCCTT GCTTAATCAT TACACCTTTG GACTGCTTCT GGGAAGGGGC AAAGCTACAG 780 TCCGGGACAG CATACCTCCT AGGTAAGCCT CCTTTACGGT GGACAAACTT TGACCCCTTG 840 GAATTCCTAG AAGAGTTAAA GAAAATAAAC TACCAAGTGG ACAGCTGGGA GGAAATGCTG 900 AATAAAGCCG AAGTTGGCCA TGGGTACATG GACCGGCCTT GCCTCAACCC AGCCGACCCA 960 GATTGCCCTG CCACAGCCCC TAACAAAAAT TCAACCAAAC CTCTTGATGT GGCCCTTGTT 1020 TTGAATGGTG GATGTCAAGG TTTATCCAGG AAGTATATGC ATTGGCAGGA GGAGTTGATT 1080 GTGGGTGGTA CCGTCAAGAA TGCCACTGGA AAACTTGTCA GCGCTCACGC CCTGCAAACC 1140 ATGTTCCAGT TAATGACTCC CAAGCAAATG TATGAACACT TCAGGGGCTA CGACTATGTC 1200 TCTCACATCA ACTGGAATGA AGACAGGGCA GCCGCCATCC TGGAGGCCTG GCAGAGGACT 1260 TACGTGGAGG TGGTTCATCA AAGTGTCGCC CCAAACTCCA CTCAAAAGGT GCTTCCCTTC 1320 ACAACCACGA CCCTGGACGA CATCCTAAAA TCCTTCTCTG ATGTCAGTGT CATCCGAGTG 1380 GCCAGCGGCT ACCTACTGAT GCTTGCCTAT GCCTGTTTAA CCATGCTGCG CTGGGACTGC 1440 TCCAAGTCCC AGGGTGCCGT GGGGCTGGCT GGCGTCCTGT TGGTTGCGCT GTCAGTGGCT 1500 GCAGGATTGG GCCTCTGCTC CTTGATTGGC ATTTCTTTTA ATGCTGCGAC AACTCAGGTT 1560 TTGCCGTTTC TTGCTCTTGG TGTTGGTGTG GATGATGTCT TCCTCCTGGC CCATGCATTC 1620 AGTGAAACAG GACAGAATAA GAGGATTCCA TTTGAGGACA GGACTGGGGA GTGCCTCAAG 1680 CGCACCGGAG CCAGCGTGGC CCTCACCTCC ATCAGCAATG TCACCGCCTT CTTCATGGCC 1740 GCATTGATCC CTATCCCTGC CCTGCGAGCG TTCTCCCTCC AGGCTGCTGT GGTGGTGGTA 1800 TTCAATTTTG CTATGGTTCT GCTCATTTTT CCTGCAATTC TCAGCATGGA TTTATACAGA 1860 CGTGAGGACA GAAGATTGGA TATTTTCTGC TGTTTCACAA GCCCCTGTGT CAGCAGGGTG 1920 ATTCAAGTTG AGCCACAGGC CTACACAGAG CCTCACAGTA ACACCCGGTA CAGCCCCCCA 1980 CCCCCATACA CCAGCCACAG CTTCGCCCAC GAAACCCATA TCACTATGCA GTCCACCGTT 2040 CAGCTCCGCA CAGAGTATGA CCCTCACACG CACGTGTACT ACACCACCGC CGAGCCACGC 2100 TCTGAGATCT CTGTACAGCC TGTTACCGTC ACCCAGGACA ACCTCAGCTG TCAGAGTCCC 2160 GAGAGCACCA GCTCTACCAG GGACCTGCTC TCCCAGTTCT CAGACTCCAG CCTCCACTGC 2220 CTCGAGCCCC CCTGCACCAA GTGGACACTC TCTTCGTTTG CAGAGAAGCA CTATGCTCCT 2280 TTCCTCCTGA AACCCAAAGC CAAGGTTGTG GTAATCCTTC TTTTCCTGGG CTTGCTGGGG 2340 GTCAGCCTTT ATGGGACCAC CCGAGTGAGA GACGGGCTGG ACCTCACGGA CATTGTTCCC 2400 CGGGAAACCA GAGAATATGA CTTCATAGCT GCCCAGTTCA AGTACTTCTC TTTCTACAAC 2460 ATGTATATAG TCACCCAGAA AGCAGACTAC CCGAATATCC AGCACCTACT TTACGACCTT 2520 CATAAGAGTT TCAGCAATGT GAAGTATGTC ATGCTGGAGG AGAACAAGCA ACTTCCCCAA 2580 ATGTGGCTGC ACTACTTTAG AGACTGGCTT CAAGGACTTC AGGATGCATT TGACAGTGAC 2640 TGGGAAACTG GGAGGATCAT GCCAAACAAT TATAAAAATG GATCAGATGA CGGGGTCCTC 2700 GCTTACAAAC TCCTGGTGCA GACTGGCAGC CGAGACAAGC CCATCGACAT TAGTCAGTTG 2760 ACTAAACAGC GTCTGGTAGA CGCAGATGGC ATCATTAATC CGAGCGCTTT CTACATCTAC 2820 CTGACCGCTT GGGTCAGCAA CGACCCTGTA GCTTACGCTG CCTCCCAGGC CAACATCCGG 2880 CCTCACCGGC CGGAGTGGGT CCATGACAAA GCCGACTACA TGCCAGAGAC CAGGCTGAGA 2940 ATCCCAGCAG CAGAGCCCAT CGAGTACGCT CAGTTCCCTT TCTACCTCAA CGGCCTACGA 3000 GACACCTCAG ACTTTGTGGA AGCCATAGAA AAAGTGAGAG TCATCTGTAA CAACTATACG 3060 AGCCTGGGAC TGTCCAGCTA CCCCAATGGC TACCCCTTCC TGTTCTGGGA GCAATACATC 3120 AGCCTGCGCC ACTGGCTGCT GCTATCCATC AGCGTGGTGC TGGCCTGCAC GTTTCTAGTG 3180 TGCGCAGTCT TCCTCCTGAA CCCCTGGACG GCCGGGATCA TTGTCATGGT CCTGGCTCTG 3240 ATGACCGTTG AGCTCTTTGG CATGATGGGC CTCATTGGGA TCAAGCTGAG TGCTGTGCCT 3300 GTGGTCATCC TGATTGCATC TGTTGGCATC GGAGTGGAGT TCACCGTCCA CGTGGCTTTG 3360 GCCTTTCTGA CAGCCATTGG GGACAAGAAC CACAGGGCTA TGCTCGCTCT GGAACACATG 3420 TTTGCTCCCG TTCTGGACGG TGCTGTGTCC ACTCTGCTGG GTGTACTGAT GCTTGCAGGG 3480 TCCGAATTTG ATTTCATTGT CAGATACTTC TTTGCCGTCC TGGCCATTCT CACCGTCTTG 3540 GGGGTTCTCA ATGGACTGGT TCTGCTGCCT GTCCTCTTAT CCTTCTTTGG ACCGTGTCCT 3600 GAGGTGTCTC CAGCCAATGG CCTAAACCGA CTGCCCACTC CTTCGCCTGA GCCGCCTCCA 3660 AGTGTCGTCC GGTTTGCCGT GCCTCCTGGT CACACGAACA ATGGGTCTGA TTCCTCCGAC 3720 TCGGAGTACA GCTCTCAGAC CACGGTGTCT GGCATCAGTG AGGAGCTCAG GCAATACGAA 3780 GCACAGCAGG GTGCCGGAGG CCCTGCCCAC CAAGTGATTG TGGAAGCCAC AGAAAACCCT 3840 GTCTTTGCCC GGTCCACTGT GGTCCATCCG GACTCCAGAC ATCAGCCTCC CTTGACCCCT 3900 CGGCAACAGC CCCACCTGGA CTCTGGCTCC TTGTCCCCTG GACGGCAAGG CCAGCAGCCT 3960 CGAAGGGATC CCCCTAGAGA AGGCTTGCGG CCACCCCCCT ACAGACCGCG CAGAGACGCT 4020 TTTGAAATTT CTACTGAAGG GCATTCTGGC CCTAGCAATA GGGACCGCTC AGGGCCCCGT 4080 GGGGCCCGTT CTCACAACCC TCGGAACCCA ACGTCCACCG CCATGGGCAG CTCTGTGCCC 4140 AGCTACTGCC AGCCCATCAC CACTGTGACG GCTTCTGCTT CGGTGACTGT TGCTGTGCAT 4200 CCCCCGCCTG GACCTGGGCG CAACCCCCGA GGGGGGCCCT GTCCAGGCTA TGAGAGCTAC 4260 CCTGAGACTG ATCACGGGGT ATTTGAGGAT CCTCATGTGC CTTTTCATGT CAGGTGTGAG 4320 AGGAGGGACT CAAAGGTGGA GGTCATAGAG CTACAGGACG TGGAATGTGA GGAGAGGCCG 4380 TGGGGGAGCA GCTCCAACTG AGGGTAATTA AAATCTGAAG CAAAGAGGCC AAAGATTGGA 4440 AAGCCCCGCC CCCACCTCTT TCCAGAACTG CTTGAAGAGA ACTGCTTGGA ATTATGGGAA 4500 GGCAGTTCAT TGTTACTGTA ACTGATTGTA TTATTKKGTG AAATATTTCT ATAAATATTT 4560 AARAGGTGTA CACATGTAAT ATACATGGAA ATGCTGTACA GTCTATTTCC TGGGGCCTCT 4620 CCACTCCTGC CCCAGAGTGG GGAGACCACA GGGGCCCTTT CCCCTGTGTA CATTGGTCTC 4680 TGTGCCACAA CCAAGCTTAA CTTAGTTTTA AAAAAAATCT CCCAGCATAT GTCGCTGCTG 4740 CTTAAATATT GTATAATTTA CTTGTATAAT TCTATGCAAA TATTGCTTAT GTAATAGGAT 4800 TATTTGTAAA GGTTTCTGTT TAAAATATTT TAAATTTGCA TATCACAACC CTGTGGTAGG 4860 ATGAATTGTT ACTGTTAACT TTTGAACACG CTATGCGTGG TAATTGTTTA ACGAGCAGAC 4920 ATGAAGAAAA CAGGTTAATC CCAGTGGCTT CTCTAGGGGT AGTTGTATAT GGTTCGCATG 4980 GGTGGATGTG TGTGTGCATG TGACTTTCCA ATGTACTGTA TTGTGGTTTG TTGTTGTTGT 5040 TGCTGTTGTT GTTCATTTTG GTGTTTTTGG TTGCTTTGTA TGATCTTAGC TCTGGCCTAG 5100 GTGGGCTGGG AAGGTCCAGG TCTTTTTCTG TCGTGATGCT GGTGGAAAGG TGACCCCAAT 5160 CATCTGTCCT ATTCTCTGGG ACTATTC 5187 1311 amino acids amino acid single linear protein 4 Met Val Ala Pro Asp Ser Glu Ala Pro Ser Asn Pro Arg Ile Thr Ala 1 5 10 15 Ala His Glu Ser Pro Cys Ala Thr Glu Ala Arg His Ser Ala Asp Leu 20 25 30 Tyr Ile Arg Thr Ser Trp Val Asp Ala Ala Leu Ala Leu Ser Glu Leu 35 40 45 Glu Lys Gly Asn Ile Glu Gly Gly Arg Thr Ser Leu Trp Ile Arg Ala 50 55 60 Trp Leu Gln Glu Gln Leu Phe Ile Leu Gly Cys Phe Leu Gln Gly Asp 65 70 75 80 Ala Gly Lys Val Leu Phe Val Ala Ile Leu Val Leu Ser Thr Phe Cys 85 90 95 Val Gly Leu Lys Ser Ala Gln Ile His Thr Arg Val Asp Gln Leu Trp 100 105 110 Val Gln Glu Gly Gly Arg Leu Glu Ala Glu Leu Lys Tyr Thr Ala Gln 115 120 125 Ala Leu Gly Glu Ala Asp Ser Ser Thr His Gln Leu Val Ile Gln Thr 130 135 140 Ala Lys Asp Pro Asp Val Ser Leu Leu His Pro Gly Ala Leu Leu Glu 145 150 155 160 His Leu Lys Val Val His Ala Ala Thr Arg Val Thr Val His Met Tyr 165 170 175 Asp Ile Glu Trp Arg Leu Lys Asp Leu Cys Tyr Ser Pro Ser Ile Pro 180 185 190 Asp Phe Glu Gly Tyr His His Ile Glu Ser Ile Ile Asp Asn Val Ile 195 200 205 Pro Cys Ala Ile Ile Thr Pro Leu Asp Cys Phe Trp Glu Gly Ser Lys 210 215 220 Leu Leu Gly Pro Asp Tyr Pro Ile Tyr Val Pro His Leu Lys His Lys 225 230 235 240 Leu Gln Trp Thr His Leu Asn Pro Leu Glu Val Val Glu Glu Val Lys 245 250 255 Lys Leu Lys Phe Gln Phe Pro Leu Ser Thr Ile Glu Ala Tyr Met Lys 260 265 270 Arg Ala Gly Ile Thr Ser Ala Tyr Met Lys Lys Pro Cys Leu Asp Pro 275 280 285 Thr Asp Pro His Cys Pro Ala Thr Ala Pro Asn Lys Lys Ser Gly His 290 295 300 Ile Pro Asp Val Ala Ala Glu Leu Ser His Gly Cys Tyr Gly Phe Ala 305 310 315 320 Ala Ala Tyr Met His Trp Pro Glu Gln Leu Ile Val Gly Gly Ala Thr 325 330 335 Arg Asn Ser Thr Ser Ala Leu Arg Lys Ala Arg Xaa Leu Gln Thr Val 340 345 350 Val Gln Leu Met Gly Glu Arg Glu Met Tyr Glu Tyr Trp Ala Asp His 355 360 365 Tyr Lys Val His Gln Ile Gly Trp Asn Gln Glu Lys Ala Ala Ala Val 370 375 380 Leu Asp Ala Trp Gln Arg Lys Phe Ala Ala Glu Val Arg Lys Ile Thr 385 390 395 400 Thr Ser Gly Ser Val Ser Ser Ala Tyr Ser Phe Tyr Pro Phe Ser Thr 405 410 415 Ser Thr Leu Asn Asp Ile Leu Gly Lys Phe Ser Glu Val Ser Leu Lys 420 425 430 Asn Ile Ile Leu Gly Tyr Met Phe Met Leu Ile Tyr Val Ala Val Thr 435 440 445 Leu Ile Gln Trp Arg Asp Pro Ile Arg Ser Gln Ala Gly Val Gly Ile 450 455 460 Ala Gly Val Leu Leu Leu Ser Ile Thr Val Ala Ala Gly Leu Gly Phe 465 470 475 480 Cys Ala Leu Leu Gly Ile Pro Phe Asn Ala Ser Ser Thr Gln Ile Val 485 490 495 Pro Phe Leu Ala Leu Gly Leu Gly Val Gln Asp Met Phe Leu Leu Thr 500 505 510 His Thr Tyr Val Glu Gln Ala Gly Asp Val Pro Arg Glu Glu Arg Thr 515 520 525 Gly Leu Val Leu Lys Lys Ser Gly Leu Ser Val Leu Leu Ala Ser Leu 530 535 540 Cys Asn Val Met Ala Phe Leu Ala Ala Ala Leu Leu Pro Ile Pro Ala 545 550 555 560 Phe Arg Val Phe Cys Leu Gln Ala Ala Ile Leu Leu Leu Phe Asn Leu 565 570 575 Gly Ser Ile Leu Leu Val Phe Pro Ala Met Ile Ser Leu Asp Leu Arg 580 585 590 Arg Arg Ser Ala Ala Arg Ala Asp Leu Leu Cys Cys Leu Met Pro Glu 595 600 605 Ser Pro Leu Pro Lys Lys Lys Ile Pro Glu Arg Ala Lys Thr Arg Lys 610 615 620 Asn Asp Lys Thr His Arg Ile Asp Thr Thr Arg Gln Pro Leu Asp Pro 625 630 635 640 Asp Val Ser Glu Asn Val Thr Lys Thr Cys Cys Leu Ser Val Ser Leu 645 650 655 Thr Lys Trp Ala Lys Asn Gln Tyr Ala Pro Phe Ile Met Arg Pro Ala 660 665 670 Val Lys Val Thr Ser Met Leu Ala Leu Ile Ala Val Ile Leu Thr Ser 675 680 685 Val Trp Gly Ala Thr Lys Val Lys Asp Gly Leu Asp Leu Thr Asp Ile 690 695 700 Val Pro Glu Asn Thr Asp Glu His Glu Phe Leu Ser Arg Gln Glu Lys 705 710 715 720 Tyr Phe Gly Phe Tyr Asn Met Tyr Ala Val Thr Gln Gly Asn Phe Glu 725 730 735 Tyr Pro Thr Asn Gln Lys Leu Leu Tyr Glu Tyr His Asp Gln Phe Val 740 745 750 Arg Ile Pro Asn Ile Ile Lys Asn Asp Asn Gly Gly Leu Thr Lys Phe 755 760 765 Trp Leu Ser Leu Phe Arg Asp Trp Leu Leu Asp Leu Gln Val Ala Phe 770 775 780 Asp Lys Glu Val Ala Ser Gly Cys Ile Thr Gln Glu Tyr Trp Cys Lys 785 790 795 800 Asn Ala Ser Asp Glu Gly Ile Leu Ala Tyr Lys Leu Met Val Gln Thr 805 810 815 Gly His Val Asp Asn Pro Ile Asp Lys Ser Leu Ile Thr Ala Gly His 820 825 830 Arg Leu Val Asp Lys Asp Gly Ile Ile Asn Pro Lys Ala Phe Tyr Asn 835 840 845 Tyr Leu Ser Ala Trp Ala Thr Asn Asp Ala Leu Ala Tyr Gly Ala Ser 850 855 860 Gln Gly Asn Leu Lys Pro Gln Pro Gln Arg Trp Ile His Ser Pro Glu 865 870 875 880 Asp Val His Leu Glu Ile Lys Lys Ser Ser Pro Leu Ile Tyr Thr Gln 885 890 895 Leu Pro Phe Tyr Leu Ser Gly Leu Ser Asp Thr Xaa Ser Ile Lys Thr 900 905 910 Leu Ile Arg Ser Val Arg Asp Leu Cys Leu Lys Tyr Glu Ala Lys Gly 915 920 925 Leu Pro Asn Phe Pro Ser Gly Ile Pro Phe Leu Phe Trp Glu Gln Tyr 930 935 940 Leu Tyr Leu Arg Thr Ser Leu Leu Leu Ala Leu Ala Cys Ala Leu Ala 945 950 955 960 Ala Val Phe Ile Ala Val Met Val Leu Leu Leu Asn Ala Trp Ala Ala 965 970 975 Val Leu Val Thr Leu Ala Leu Ala Thr Leu Val Leu Gln Leu Leu Gly 980 985 990 Val Met Ala Leu Leu Gly Val Lys Leu Ser Ala Met Pro Ala Val Leu 995 1000 1005 Leu Val Leu Ala Ile Gly Arg Gly Val His Phe Thr Val His Leu Cys 1010 1015 1020 Leu Gly Phe Val Thr Ser Ile Gly Cys Lys Arg Arg Arg Ala Ser Leu 1025 1030 1035 1040 Ala Leu Glu Ser Val Leu Ala Pro Val Val His Gly Ala Leu Ala Ala 1045 1050 1055 Ala Leu Ala Ala Ser Met Leu Ala Ala Ser Glu Cys Gly Phe Val Ala 1060 1065 1070 Arg Leu Phe Leu Arg Leu Leu Leu Asp Ile Val Phe Leu Gly Leu Ile 1075 1080 1085 Asp Gly Leu Leu Phe Phe Pro Ile Val Leu Ser Ile Leu Gly Pro Ala 1090 1095 1100 Ala Glu Val Arg Pro Ile Glu His Pro Glu Arg Leu Ser Thr Pro Ser 1105 1110 1115 1120 Pro Lys Cys Ser Pro Ile His Pro Arg Lys Ser Ser Ser Ser Ser Gly 1125 1130 1135 Gly Gly Asp Lys Ser Ser Arg Thr Ser Lys Ser Ala Pro Arg Pro Cys 1140 1145 1150 Ala Pro Ser Leu Thr Thr Ile Thr Glu Glu Pro Ser Ser Trp His Ser 1155 1160 1165 Ser Ala His Ser Val Gln Ser Ser Met Gln Ser Ile Val Val Gln Pro 1170 1175 1180 Glu Val Val Val Glu Thr Thr Thr Tyr Asn Gly Ser Asp Ser Ala Ser 1185 1190 1195 1200 Gly Arg Ser Thr Pro Thr Lys Ser Ser His Gly Gly Ala Ile Thr Thr 1205 1210 1215 Thr Lys Val Thr Ala Thr Ala Asn Ile Lys Val Glu Val Val Thr Pro 1220 1225 1230 Ser Asp Arg Lys Ser Arg Arg Ser Tyr His Tyr Tyr Asp Arg Arg Arg 1235 1240 1245 Asp Arg Asp Glu Asp Arg Asp Arg Asp Arg Glu Arg Asp Arg Asp Arg 1250 1255 1260 Asp Arg Asp Arg Asp Arg Asp Arg Asp Arg Asp Arg Asp Arg Asp Arg 1265 1270 1275 1280 Glu Arg Ser Arg Glu Arg Asp Arg Arg Asp Arg Tyr Arg Asp Glu Arg 1285 1290 1295 Asp His Arg Ala Ser Pro Arg Glu Lys Arg Gln Arg Phe Trp Thr 1300 1305 1310 4434 base pairs nucleic acid single linear cDNA 5 CGAAACAAGA GAGCGAGTGA GAGTAGGGAG AGCGTCTGTG TTGTGTGTTG AGTGTCGCCC 60 ACGCACACAG GCGCAAAACA GTGCACACAG ACGCCCGCTG GGCAAGAGAG AGTGAGAGAG 120 AGAAACAGCG GCGCGCGCTC GCCTAATGAA GTTGTTGGCC TGGCTGGCGT GCCGCATCCA 180 CGAGATACAG ATACATCTCT CATGGACCGC GACAGCCTCC CACGCGTTCC GGACACACAC 240 GGCGATGTGG TCGATGAGAA ATTATTCTCG GATCTTTACA TACGCACCAG CTGGGTGGAC 300 GCCCAAGTGG CGCTCGATCA GATAGATAAG GGCAAAGCGC GTGGCAGCCG CACGGCGATC 360 TATCTGCGAT CAGTATTCCA GTCCCACCTC GAAACCCTCG GCAGCTCCGT GCAAAAGCAC 420 GCGGGCAAGG TGCTATTCGT GGCTATCCTG GTGCTGAGCA CCTTCTGCGT CGGCCTGAAG 480 AGCGCCCAGA TCCACTCCAA GGTGCACCAG CTGTGGATCC AGGAGGGCGG CCGGCTGGAG 540 GCGGAACTGG CCTACACACA GAAGACGATC GGCGAGGACG AGTCGGCCAC GCATCAGCTG 600 CTCATTCAGA CGACCCACGA CCCGAACGCC TCCGTCCTGC ATCCGCAGGC GCTGCTTGCC 660 CACCTGGAGG TCCTGGTCAA GGCCACCGCC GTCAAGGTGC ACCTCTACGA CACCGAATGG 720 GGGCTGCGCG ACATGTGCAA CATGCCGAGC ACGCCCTCCT TCGAGGGCAT CTACTACATC 780 GAGCAGATCC TGCGCCACCT CATTCCGTGC TCGATCATCA CGCCGCTGGA CTGTTTCTGG 840 GAGGGAAGCC AGCTGTTGGG TCCGGAATCA GCGGTCGTTA TACCAGGCCT CAACCAACGA 900 CTCCTGTGGA CCACCCTGAA TCCCGCCTCT GTGATGCAGT ATATGAAACA AAAGATGTCC 960 GAGGAAAAGA TCAGCTTCGA CTTCGAGACC GTGGAGCAGT ACATGAAGCG TGCGGCCATT 1020 GGCAGTGGCT ACATGGAGAA GCCCTGCCTG AACCCACTGA ATCCCAATTG CCCGGACACG 1080 GCACCGAACA AGAACAGCAC CCAGCCGCCG GATGTGGGAG CCATCCTGTC CGGAGGCTGC 1140 TACGGTTATG CCGCGAAGCA CATGCACTGG CCGGAGGAGC TGATTGTGGG CGGACGGAAG 1200 AGGAACCGCA GCGGACACTT GAGGAAGGCC CAGGCCCTGC AGTCGGTGGT GCAGCTGATG 1260 ACCGAGAAGG AAATGTACGA CCAGTGGCAG GACAACTACA AGGTGCACCA TCTTGGATGG 1320 ACGCAGGAGA AGGCAGCGGA GGTTTTGAAC GCCTGGCAGC GCAACTTTTC GCGGGAGGTG 1380 GAACAGCTGC TACGTAAACA GTCGAGAATT GCCACCAACT ACGATATCTA CGTGTTCAGC 1440 TCGGCTGCAC TGGATGACAT CCTGGCCAAG TTCTCCCATC CCAGCGCCTT GTCCATTGTC 1500 ATCGGCGTGG CCGTCACCGT TTTGTATGCC TTTTGCACGC TCCTCCGCTG GAGGGACCCC 1560 GTCCGTGGCC AGAGCAGTGT GGGCGTGGCC GGAGTTCTGC TCATGTGCTT CAGTACCGCC 1620 GCCGGATTGG GATTGTCAGC CCTGCTCGGT ATCGTTTTCA ATGCGCTGAC CGCTGCCTAT 1680 GCGGAGAGCA ATCGGCGGGA GCAGACCAAG CTGATTCTCA AGAACGCCAG CACCCAGGTG 1740 GTTCCGTTTT TGGCCCTTGG TCTGGGCGTC GATCACATCT TCATAGTGGG ACCGAGCATC 1800 CTGTTCAGTG CCTGCAGCAC CGCAGGATCC TTCTTTGCGG CCGCCTTTAT TCCGGTGCCG 1860 GCTTTGAAGG TATTCTGTCT GCAGGCTGCC ATCGTAATGT GCTCCAATTT GGCAGCGGCT 1920 CTATTGGTTT TTCCGGCCAT GATTTCGTTG GATCTACGGA GACGTACCGC CGGCAGGGCG 1980 GACATCTTCT GCTGCTGTTT TCCGGTGTGG AAGGAACAGC CGAAGGTGGC ACCTCCGGTG 2040 CTGCCGCTGA ACAACAACAA CGGGCGCGGG GCCCGGCATC CGAAGAGCTG CAACAACAAC 2100 AGGGTGCCGC TGCCCGCCCA GAATCCTCTG CTGGAACAGA GGGCAGACAT CCCTGGGAGC 2160 AGTCACTCAC TGGCGTCCTT CTCCCTGGCA ACCTTCGCCT TTCAGCACTA CACTCCCTTC 2220 CTCATGCGCA GCTGGGTGAA GTTCCTGACC GTTATGGGTT TCCTGGCGGC CCTCATATCC 2280 AGCTTGTATG CCTCCACGCG CCTTCAGGAT GGCCTGGACA TTATTGATCT GGTGCCCAAG 2340 GACAGCAACG AGCACAAGTT CCTGGATGCT CAAACTCGGC TCTTTGGCTT CTACAGCATG 2400 TATGCGGTTA CCCAGGGCAA CTTTGAATAT CCCACCCAGC AGCAGTTGCT CAGGGACTAC 2460 CATGATTCCT TTGTGCGGGT GCCACATGTG ATCAAGAATG ATAACGGTGG ACTGCCGGAC 2520 TTCTGGCTGC TGCTCTTCAG CGAGTGGCTG GGTAATCTGC AAAAGATATT CGACGAGGAA 2580 TACCGCGACG GACGGCTGAC CAAGGAGTGC TGGTTCCCAA ACGCCAGCAG CGATGCCATC 2640 CTGGCCTACA AGCTAATCGT GCAAACCGGC CATGTGGACA ACCCCGTGGA CAAGGAACTG 2700 GTGCTCACCA ATCGCCTGGT CAACAGCGAT GGCATCATCA ACCAACGCGC CTTCTACAAC 2760 TATCTGTCGG CATGGGCCAC CAACGACGTC TTCGCCTACG GAGCTTCTCA GGGCAAATTG 2820 TATCCGGAAC CGCGCCAGTA TTTTCACCAA CCCAACGAGT ACGATCTTAA GATACCCAAG 2880 AGTCTGCCAT TGGTCTACGC TCAGATGCCC TTTTACCTCC ACGGACTAAC AGATACCTCG 2940 CAGATCAAGA CCCTGATAGG TCATATTCGC GACCTGAGCG TCAAGTACGA GGGCTTCGGC 3000 CTGCCCAACT ATCCATCGGG CATTCCCTTC ATCTTCTGGG AGCAGTACAT GACCCTGCGC 3060 TCCTCACTGG CCATGATCCT GGCCTGCGTG CTACTCGCCG CCCTGGTGCT GGTCTCCCTG 3120 CTCCTGCTCT CCGTTTGGGC CGCCGTTCTC GTGATCCTCA GCGTTCTGGC CTCGCTGGCC 3180 CAGATCTTTG GGGCCATGAC TCTGCTGGGC ATCAAACTCT CGGCCATTCC GGCAGTCATA 3240 CTCATCCTCA GCGTGGGCAT GATGCTGTGC TTCAATGTGC TGATATCACT GGGCTTCATG 3300 ACATCCGTTG GCAACCGACA GCGCCGCGTC CAGCTGAGCA TGCAGATGTC CCTGGGACCA 3360 CTTGTCCACG GCATGCTGAC CTCCGGAGTG GCCGTGTTCA TGCTCTCCAC GTCGCCCTTT 3420 GAGTTTGTGA TCCGGCACTT CTGCTGGCTT CTGCTGGTGG TCTTATGCGT TGGCGCCTGC 3480 AACAGCCTTT TGGTGTTCCC CATCCTACTG AGCATGGTGG GACCGGAGGC GGAGCTGGTG 3540 CCGCTGGAGC ATCCAGACCG CATATCCACG CCCTCTCCGC TGCCCGTGCG CAGCAGCAAG 3600 AGATCGGGCA AATCCTATGT GGTGCAGGGA TCGCGATCCT CGCGAGGCAG CTGCCAGAAG 3660 TCGCATCACC ACCACCACAA AGACCTTAAT GATCCATCGC TGACGACGAT CACCGAGGAG 3720 CCGCAGTCGT GGAAGTCCAG CAACTCGTCC ATCCAGATGC CCAATGATTG GACCTACCAG 3780 CCGCGGGAAC AGCGACCCGC CTCCTACGCG GCCCCGCCCC CCGCCTATCA CAAGGCCGCC 3840 GCCCAGCAGC ACCACCAGCA TCAGGGCCCG CCCACAACGC CCCCGCCTCC CTTCCCGACG 3900 GCCTATCCGC CGGAGCTGCA GAGCATCGTG GTGCAGCCGG AGGTGACGGT GGAGACGACG 3960 CACTCGGACA GCAACACCAC CAAGGTGACG GCCACGGCCA ACATCAAGGT GGAGCTGGCC 4020 ATGCCCGGCA GGGCGGTGCG CAGCTATAAC TTTACGAGTT AGCACTAGCA CTAGTTCCTG 4080 TAGCTATTAG GACGTATCTT TAGACTCTAG CCTAAGCCGT AACCCTATTT GTATCTGTAA 4140 AATCGATTTG TCCAGCGGGT CTGCTGAGGA TTTCGTTCTC ATGGATTCTC ATGGATTCTC 4200 ATGGATGCTT AAATGGCATG GTAATTGGCA AAATATCAAT TTTTGTGTCT CAAAAAGATG 4260 CATTAGCTTA TGGTTTCAAG ATACATTTTT AAAGAGTCCG CCAGATATTT ATATAAAAAA 4320 AATCCAAAAT CGACGTATCC ATGAAAATTG AAAAGCTAAG CAGACCCGTA TGTATGTATA 4380 TGTGTATGCA TGTTAGTTAA TTTCCCGAAG TCCGGTATTT ATAGCAGCTG CCTT 4434 1285 amino acids amino acid single linear protein 6 Met Asp Arg Asp Ser Leu Pro Arg Val Pro Asp Thr His Gly Asp Val 1 5 10 15 Val Asp Glu Lys Leu Phe Ser Asp Leu Tyr Ile Arg Thr Ser Trp Val 20 25 30 Asp Ala Gln Val Ala Leu Asp Gln Ile Asp Lys Gly Lys Ala Arg Gly 35 40 45 Ser Arg Thr Ala Ile Tyr Leu Arg Ser Val Phe Gln Ser His Leu Glu 50 55 60 Thr Leu Gly Ser Ser Val Gln Lys His Ala Gly Lys Val Leu Phe Val 65 70 75 80 Ala Ile Leu Val Leu Ser Thr Phe Cys Val Gly Leu Lys Ser Ala Gln 85 90 95 Ile His Ser Lys Val His Gln Leu Trp Ile Gln Glu Gly Gly Arg Leu 100 105 110 Glu Ala Glu Leu Ala Tyr Thr Gln Lys Thr Ile Gly Glu Asp Glu Ser 115 120 125 Ala Thr His Gln Leu Leu Ile Gln Thr Thr His Asp Pro Asn Ala Ser 130 135 140 Val Leu His Pro Gln Ala Leu Leu Ala His Leu Glu Val Leu Val Lys 145 150 155 160 Ala Thr Ala Val Lys Val His Leu Tyr Asp Thr Glu Trp Gly Leu Arg 165 170 175 Asp Met Cys Asn Met Pro Ser Thr Pro Ser Phe Glu Gly Ile Tyr Tyr 180 185 190 Ile Glu Gln Ile Leu Arg His Leu Ile Pro Cys Ser Ile Ile Thr Pro 195 200 205 Leu Asp Cys Phe Trp Glu Gly Ser Gln Leu Leu Gly Pro Glu Ser Ala 210 215 220 Val Val Ile Pro Gly Leu Asn Gln Arg Leu Leu Trp Thr Thr Leu Asn 225 230 235 240 Pro Ala Ser Val Met Gln Tyr Met Lys Gln Lys Met Ser Glu Glu Lys 245 250 255 Ile Ser Phe Asp Phe Glu Thr Val Glu Gln Tyr Met Lys Arg Ala Ala 260 265 270 Ile Gly Ser Gly Tyr Met Glu Lys Pro Cys Leu Asn Pro Leu Asn Pro 275 280 285 Asn Cys Pro Asp Thr Ala Pro Asn Lys Asn Ser Thr Gln Pro Pro Asp 290 295 300 Val Gly Ala Ile Leu Ser Gly Gly Cys Tyr Gly Tyr Ala Ala Lys His 305 310 315 320 Met His Trp Pro Glu Glu Leu Ile Val Gly Gly Arg Lys Arg Asn Arg 325 330 335 Ser Gly His Leu Arg Lys Ala Gln Ala Leu Gln Ser Val Val Gln Leu 340 345 350 Met Thr Glu Lys Glu Met Tyr Asp Gln Trp Gln Asp Asn Tyr Lys Val 355 360 365 His His Leu Gly Trp Thr Gln Glu Lys Ala Ala Glu Val Leu Asn Ala 370 375 380 Trp Gln Arg Asn Phe Ser Arg Glu Val Glu Gln Leu Leu Arg Lys Gln 385 390 395 400 Ser Arg Ile Ala Thr Asn Tyr Asp Ile Tyr Val Phe Ser Ser Ala Ala 405 410 415 Leu Asp Asp Ile Leu Ala Lys Phe Ser His Pro Ser Ala Leu Ser Ile 420 425 430 Val Ile Gly Val Ala Val Thr Val Leu Tyr Ala Phe Cys Thr Leu Leu 435 440 445 Arg Trp Arg Asp Pro Val Arg Gly Gln Ser Ser Val Gly Val Ala Gly 450 455 460 Val Leu Leu Met Cys Phe Ser Thr Ala Ala Gly Leu Gly Leu Ser Ala 465 470 475 480 Leu Leu Gly Ile Val Phe Asn Ala Leu Thr Ala Ala Tyr Ala Glu Ser 485 490 495 Asn Arg Arg Glu Gln Thr Lys Leu Ile Leu Lys Asn Ala Ser Thr Gln 500 505 510 Val Val Pro Phe Leu Ala Leu Gly Leu Gly Val Asp His Ile Phe Ile 515 520 525 Val Gly Pro Ser Ile Leu Phe Ser Ala Cys Ser Thr Ala Gly Ser Phe 530 535 540 Phe Ala Ala Ala Phe Ile Pro Val Pro Ala Leu Lys Val Phe Cys Leu 545 550 555 560 Gln Ala Ala Ile Val Met Cys Ser Asn Leu Ala Ala Ala Leu Leu Val 565 570 575 Phe Pro Ala Met Ile Ser Leu Asp Leu Arg Arg Arg Thr Ala Gly Arg 580 585 590 Ala Asp Ile Phe Cys Cys Cys Phe Pro Val Trp Lys Glu Gln Pro Lys 595 600 605 Val Ala Pro Pro Val Leu Pro Leu Asn Asn Asn Asn Gly Arg Gly Ala 610 615 620 Arg His Pro Lys Ser Cys Asn Asn Asn Arg Val Pro Leu Pro Ala Gln 625 630 635 640 Asn Pro Leu Leu Glu Gln Arg Ala Asp Ile Pro Gly Ser Ser His Ser 645 650 655 Leu Ala Ser Phe Ser Leu Ala Thr Phe Ala Phe Gln His Tyr Thr Pro 660 665 670 Phe Leu Met Arg Ser Trp Val Lys Phe Leu Thr Val Met Gly Phe Leu 675 680 685 Ala Ala Leu Ile Ser Ser Leu Tyr Ala Ser Thr Arg Leu Gln Asp Gly 690 695 700 Leu Asp Ile Ile Asp Leu Val Pro Lys Asp Ser Asn Glu His Lys Phe 705 710 715 720 Leu Asp Ala Gln Thr Arg Leu Phe Gly Phe Tyr Ser Met Tyr Ala Val 725 730 735 Thr Gln Gly Asn Phe Glu Tyr Pro Thr Gln Gln Gln Leu Leu Arg Asp 740 745 750 Tyr His Asp Ser Phe Arg Val Pro His Val Ile Lys Asn Asp Asn Gly 755 760 765 Gly Leu Pro Asp Phe Trp Leu Leu Leu Phe Ser Glu Trp Leu Gly Asn 770 775 780 Leu Gln Lys Ile Phe Asp Glu Glu Tyr Arg Asp Gly Arg Leu Thr Lys 785 790 795 800 Glu Cys Trp Phe Pro Asn Ala Ser Ser Asp Ala Ile Leu Ala Tyr Lys 805 810 815 Leu Ile Val Gln Thr Gly His Val Asp Asn Pro Val Asp Lys Glu Leu 820 825 830 Val Leu Thr Asn Arg Leu Val Asn Ser Asp Gly Ile Ile Asn Gln Arg 835 840 845 Ala Phe Tyr Asn Tyr Leu Ser Ala Trp Ala Thr Asn Asp Val Phe Ala 850 855 860 Tyr Gly Ala Ser Gln Gly Lys Leu Tyr Pro Glu Pro Arg Gln Tyr Phe 865 870 875 880 His Gln Pro Asn Glu Tyr Asp Leu Lys Ile Pro Lys Ser Leu Pro Leu 885 890 895 Val Tyr Ala Gln Met Pro Phe Tyr Leu His Gly Leu Thr Asp Thr Ser 900 905 910 Gln Ile Lys Thr Leu Ile Gly His Ile Arg Asp Leu Ser Val Lys Tyr 915 920 925 Glu Gly Phe Gly Leu Pro Asn Tyr Pro Ser Gly Ile Pro Phe Ile Phe 930 935 940 Trp Glu Gln Tyr Met Thr Leu Arg Ser Ser Leu Ala Met Ile Leu Ala 945 950 955 960 Cys Val Leu Leu Ala Ala Leu Val Leu Val Ser Leu Leu Leu Leu Ser 965 970 975 Val Trp Ala Ala Val Leu Val Ile Leu Ser Val Leu Ala Ser Leu Ala 980 985 990 Gln Ile Phe Gly Ala Met Thr Leu Leu Gly Ile Lys Leu Ser Ala Ile 995 1000 1005 Pro Ala Val Ile Leu Ile Leu Ser Val Gly Met Met Leu Cys Phe Asn 1010 1015 1020 Val Leu Ile Ser Leu Gly Phe Met Thr Ser Val Gly Asn Arg Gln Arg 1025 1030 1035 1040 Arg Val Gln Leu Ser Met Gln Met Ser Leu Gly Pro Leu Val His Gly 1045 1050 1055 Met Leu Thr Ser Gly Val Ala Val Phe Met Leu Ser Thr Ser Pro Phe 1060 1065 1070 Glu Phe Val Ile Arg His Phe Cys Trp Leu Leu Leu Val Val Leu Cys 1075 1080 1085 Val Gly Ala Cys Asn Ser Leu Leu Val Phe Pro Ile Leu Leu Ser Met 1090 1095 1100 Val Gly Pro Glu Ala Glu Leu Val Pro Leu Glu His Pro Asp Arg Ile 1105 1110 1115 1120 Ser Thr Pro Ser Pro Leu Pro Val Arg Ser Ser Lys Arg Ser Gly Lys 1125 1130 1135 Ser Tyr Val Val Gln Gly Ser Arg Ser Ser Arg Gly Ser Cys Gln Lys 1140 1145 1150 Ser His His His His His Lys Asp Leu Asn Asp Pro Ser Leu Thr Thr 1155 1160 1165 Ile Thr Glu Glu Pro Gln Ser Trp Lys Ser Ser Asn Ser Ser Ile Gln 1170 1175 1180 Met Pro Asn Asp Trp Thr Tyr Gln Pro Arg Glu Gln Arg Pro Ala Ser 1185 1190 1195 1200 Tyr Ala Ala Pro Pro Pro Ala Tyr His Lys Ala Ala Ala Gln Gln His 1205 1210 1215 His Gln His Gln Gly Pro Pro Thr Thr Pro Pro Pro Pro Phe Pro Thr 1220 1225 1230 Ala Tyr Pro Pro Glu Leu Gln Ser Ile Val Val Gln Pro Glu Val Thr 1235 1240 1245 Val Glu Thr Thr His Ser Asp Ser Asn Thr Thr Lys Val Thr Ala Thr 1250 1255 1260 Ala Asn Ile Lys Val Glu Leu Ala Met Pro Gly Arg Ala Val Arg Ser 1265 1270 1275 1280 Tyr Asn Phe Thr Ser 1285 345 base pairs nucleic acid single linear DNA (genomic) 7 AAGGTCCATC AGCTTTGGAT ACAGGAAGGT GGTTCGCTCG AGCATGAGCT AGCCTACACG 60 CAGAAATCGC TCGGCGAGAT GGACTCCTCC ACGCACCAGC TGCTAATCCA AACNCCCAAA 120 GATATGGACG CCTCGATACT GCACCCGAAC GCGCTACTGA CGCACCTGGA CGTGGTGAAG 180 AAAGCGATCT CGGTGACGGT GCACATGTAC GACATCACGT GGAGNCTCAA GGACATGTGC 240 TACTCGCCCA GCATACCGAG NTTCGATACG CACTTTATCG AGCAGATCTT CGAGAACATC 300 ATACCGTGCG CGATCATCAC GCCGCTGGAT TGCTTTTGGG AGGGA 345 115 amino acids amino acid single linear peptide 8 Lys Val His Gln Leu Trp Ile Gln Glu Gly Gly Ser Leu Glu His Glu 1 5 10 15 Leu Ala Tyr Thr Gln Lys Ser Leu Gly Glu Met Asp Ser Ser Thr His 20 25 30 Gln Leu Leu Ile Gln Thr Pro Lys Asp Met Asp Ala Ser Ile Leu His 35 40 45 Pro Asn Ala Leu Leu Thr His Leu Asp Val Val Lys Lys Ala Ile Ser 50 55 60 Val Thr Val His Met Tyr Asp Ile Thr Trp Xaa Leu Lys Asp Met Cys 65 70 75 80 Tyr Ser Pro Ser Ile Pro Xaa Phe Asp Thr His Phe Ile Glu Gln Ile 85 90 95 Phe Glu Asn Ile Ile Pro Cys Ala Ile Ile Thr Pro Leu Asp Cys Phe 100 105 110 Trp Glu Gly 115 5187 base pairs nucleic acid single linear cDNA 9 GGGTCTGTCA CCCGGAGCCG GAGTCCCCGG CGGCCAGCAG CGTCCTCGCG AGCCGAGCGC 60 CCAGGCGCGC CCGGAGCCCG CGGCGGCGGC GGCAACATGG CCTCGGCTGG TAACGCCGCC 120 GGGGCCCTGG GCAGGCAGGC CGGCGGCGGG AGGCGCAGAC GGACCGGGGG ACCGCACCGC 180 GCCGCGCCGG ACCGGGACTA TCTGCACCGG CCCAGCTACT GCGACGCCGC CTTCGCTCTG 240 GAGCAGATTT CCAAGGGGAA GGCTACTGGC CGGAAAGCGC CGCTGTGGCT GAGAGCGAAG 300 TTTCAGAGAC TCTTATTTAA ACTGGGTTGT TACATTCAAA AGAACTGCGG CAAGTTTTTG 360 GTTGTGGGTC TCCTCATATT TGGGGCCTTC GCTGTGGGAT TAAAGGCAGC TAATCTCGAG 420 ACCAACGTGG AGGAGCTGTG GGTGGAAGTT GGTGGACGAG TGAGTCGAGA ATTAAATTAT 480 ACCCGTCAGA AGATAGGAGA AGAGGCTATG TTTAATCCTC AACTCATGAT ACAGACTCCA 540 AAAGAAGAAG GCGCTAATGT TCTGACCACA GAGGCTCTCC TGCAACACCT GGACTCAGCA 600 CTCCAGGCCA GTCGTGTGCA CGTCTACATG TATAACAGGC AATGGAAGTT GGAACATTTG 660 TGCTACAAAT CAGGGGAACT TATCACGGAG ACAGGTTACA TGGATCAGAT AATAGAATAC 720 CTTTACCCTT GCTTAATCAT TACACCTTTG GACTGCTTCT GGGAAGGGGC AAAGCTACAG 780 TCCGGGACAG CATACCTCCT AGGTAAGCCT CCTTTACGGT GGACAAACTT TGACCCCTTG 840 GAATTCCTAG AAGAGTTAAA GAAAATAAAC TACCAAGTGG ACAGCTGGGA GGAAATGCTG 900 AATAAAGCCG AAGTTGGCCA TGGGTACATG GACCGGCCTT GCCTCAACCC AGCCGACCCA 960 GATTGCCCTG CCACAGCCCC TAACAAAAAT TCAACCAAAC CTCTTGATGT GGCCCTTGTT 1020 TTGAATGGTG GATGTCAAGG TTTATCCAGG AAGTATATGC ATTGGCAGGA GGAGTTGATT 1080 GTGGGTGGTA CCGTCAAGAA TGCCACTGGA AAACTTGTCA GCGCTCACGC CCTGCAAACC 1140 ATGTTCCAGT TAATGACTCC CAAGCAAATG TATGAACACT TCAGGGGCTA CGACTATGTC 1200 TCTCACATCA ACTGGAATGA AGACAGGGCA GCCGCCATCC TGGAGGCCTG GCAGAGGACT 1260 TACGTGGAGG TGGTTCATCA AAGTGTCGCC CCAAACTCCA CTCAAAAGGT GCTTCCCTTC 1320 ACAACCACGA CCCTGGACGA CATCCTAAAA TCCTTCTCTG ATGTCAGTGT CATCCGAGTG 1380 GCCAGCGGCT ACCTACTGAT GCTTGCCTAT GCCTGTTTAA CCATGCTGCG CTGGGACTGC 1440 TCCAAGTCCC AGGGTGCCGT GGGGCTGGCT GGCGTCCTGT TGGTTGCGCT GTCAGTGGCT 1500 GCAGGATTGG GCCTCTGCTC CTTGATTGGC ATTTCTTTTA ATGCTGCGAC AACTCAGGTT 1560 TTGCCGTTTC TTGCTCTTGG TGTTGGTGTG GATGATGTCT TCCTCCTGGC CCATGCATTC 1620 AGTGAAACAG GACAGAATAA GAGGATTCCA TTTGAGGACA GGACTGGGGA GTGCCTCAAG 1680 CGCACCGGAG CCAGCGTGGC CCTCACCTCC ATCAGCAATG TCACCGCCTT CTTCATGGCC 1740 GCATTGATCC CTATCCCTGC CCTGCGAGCG TTCTCCCTCC AGGCTGCTGT GGTGGTGGTA 1800 TTCAATTTTG CTATGGTTCT GCTCATTTTT CCTGCAATTC TCAGCATGGA TTTATACAGA 1860 CGTGAGGACA GAAGATTGGA TATTTTCTGC TGTTTCACAA GCCCCTGTGT CAGCAGGGTG 1920 ATTCAAGTTG AGCCACAGGC CTACACAGAG CCTCACAGTA ACACCCGGTA CAGCCCCCCA 1980 CCCCCATACA CCAGCCACAG CTTCGCCCAC GAAACCCATA TCACTATGCA GTCCACCGTT 2040 CAGCTCCGCA CAGAGTATGA CCCTCACACG CACGTGTACT ACACCACCGC CGAGCCACGC 2100 TCTGAGATCT CTGTACAGCC TGTTACCGTC ACCCAGGACA ACCTCAGCTG TCAGAGTCCC 2160 GAGAGCACCA GCTCTACCAG GGACCTGCTC TCCCAGTTCT CAGACTCCAG CCTCCACTGC 2220 CTCGAGCCCC CCTGCACCAA GTGGACACTC TCTTCGTTTG CAGAGAAGCA CTATGCTCCT 2280 TTCCTCCTGA AACCCAAAGC CAAGGTTGTG GTAATCCTTC TTTTCCTGGG CTTGCTGGGG 2340 GTCAGCCTTT ATGGGACCAC CCGAGTGAGA GACGGGCTGG ACCTCACGGA CATTGTTCCC 2400 CGGGAAACCA GAGAATATGA CTTCATAGCT GCCCAGTTCA AGTACTTCTC TTTCTACAAC 2460 ATGTATATAG TCACCCAGAA AGCAGACTAC CCGAATATCC AGCACCTACT TTACGACCTT 2520 CATAAGAGTT TCAGCAATGT GAAGTATGTC ATGCTGGAGG AGAACAAGCA ACTTCCCCAA 2580 ATGTGGCTGC ACTACTTTAG AGACTGGCTT CAAGGACTTC AGGATGCATT TGACAGTGAC 2640 TGGGAAACTG GGAGGATCAT GCCAAACAAT TATAAAAATG GATCAGATGA CGGGGTCCTC 2700 GCTTACAAAC TCCTGGTGCA GACTGGCAGC CGAGACAAGC CCATCGACAT TAGTCAGTTG 2760 ACTAAACAGC GTCTGGTAGA CGCAGATGGC ATCATTAATC CGAGCGCTTT CTACATCTAC 2820 CTGACCGCTT GGGTCAGCAA CGACCCTGTA GCTTACGCTG CCTCCCAGGC CAACATCCGG 2880 CCTCACCGGC CGGAGTGGGT CCATGACAAA GCCGACTACA TGCCAGAGAC CAGGCTGAGA 2940 ATCCCAGCAG CAGAGCCCAT CGAGTACGCT CAGTTCCCTT TCTACCTCAA CGGCCTACGA 3000 GACACCTCAG ACTTTGTGGA AGCCATAGAA AAAGTGAGAG TCATCTGTAA CAACTATACG 3060 AGCCTGGGAC TGTCCAGCTA CCCCAATGGC TACCCCTTCC TGTTCTGGGA GCAATACATC 3120 AGCCTGCGCC ACTGGCTGCT GCTATCCATC AGCGTGGTGC TGGCCTGCAC GTTTCTAGTG 3180 TGCGCAGTCT TCCTCCTGAA CCCCTGGACG GCCGGGATCA TTGTCATGGT CCTGGCTCTG 3240 ATGACCGTTG AGCTCTTTGG CATGATGGGC CTCATTGGGA TCAAGCTGAG TGCTGTGCCT 3300 GTGGTCATCC TGATTGCATC TGTTGGCATC GGAGTGGAGT TCACCGTCCA CGTGGCTTTG 3360 GCCTTTCTGA CAGCCATTGG GGACAAGAAC CACAGGGCTA TGCTCGCTCT GGAACACATG 3420 TTTGCTCCCG TTCTGGACGG TGCTGTGTCC ACTCTGCTGG GTGTACTGAT GCTTGCAGGG 3480 TCCGAATTTG ATTTCATTGT CAGATACTTC TTTGCCGTCC TGGCCATTCT CACCGTCTTG 3540 GGGGTTCTCA ATGGACTGGT TCTGCTGCCT GTCCTCTTAT CCTTCTTTGG ACCGTGTCCT 3600 GAGGTGTCTC CAGCCAATGG CCTAAACCGA CTGCCCACTC CTTCGCCTGA GCCGCCTCCA 3660 AGTGTCGTCC GGTTTGCCGT GCCTCCTGGT CACACGAACA ATGGGTCTGA TTCCTCCGAC 3720 TCGGAGTACA GCTCTCAGAC CACGGTGTCT GGCATCAGTG AGGAGCTCAG GCAATACGAA 3780 GCACAGCAGG GTGCCGGAGG CCCTGCCCAC CAAGTGATTG TGGAAGCCAC AGAAAACCCT 3840 GTCTTTGCCC GGTCCACTGT GGTCCATCCG GACTCCAGAC ATCAGCCTCC CTTGACCCCT 3900 CGGCAACAGC CCCACCTGGA CTCTGGCTCC TTGTCCCCTG GACGGCAAGG CCAGCAGCCT 3960 CGAAGGGATC CCCCTAGAGA AGGCTTGCGG CCACCCCCCT ACAGACCGCG CAGAGACGCT 4020 TTTGAAATTT CTACTGAAGG GCATTCTGGC CCTAGCAATA GGGACCGCTC AGGGCCCCGT 4080 GGGGCCCGTT CTCACAACCC TCGGAACCCA ACGTCCACCG CCATGGGCAG CTCTGTGCCC 4140 AGCTACTGCC AGCCCATCAC CACTGTGACG GCTTCTGCTT CGGTGACTGT TGCTGTGCAT 4200 CCCCCGCCTG GACCTGGGCG CAACCCCCGA GGGGGGCCCT GTCCAGGCTA TGAGAGCTAC 4260 CCTGAGACTG ATCACGGGGT ATTTGAGGAT CCTCATGTGC CTTTTCATGT CAGGTGTGAG 4320 AGGAGGGACT CAAAGGTGGA GGTCATAGAG CTACAGGACG TGGAATGTGA GGAGAGGCCG 4380 TGGGGGAGCA GCTCCAACTG AGGGTAATTA AAATCTGAAG CAAAGAGGCC AAAGATTGGA 4440 AAGCCCCGCC CCCACCTCTT TCCAGAACTG CTTGAAGAGA ACTGCTTGGA ATTATGGGAA 4500 GGCAGTTCAT TGTTACTGTA ACTGATTGTA TTATTKKGTG AAATATTTCT ATAAATATTT 4560 AARAGGTGTA CACATGTAAT ATACATGGAA ATGCTGTACA GTCTATTTCC TGGGGCCTCT 4620 CCACTCCTGC CCCAGAGTGG GGAGACCACA GGGGCCCTTT CCCCTGTGTA CATTGGTCTC 4680 TGTGCCACAA CCAAGCTTAA CTTAGTTTTA AAAAAAATCT CCCAGCATAT GTCGCTGCTG 4740 CTTAAATATT GTATAATTTA CTTGTATAAT TCTATGCAAA TATTGCTTAT GTAATAGGAT 4800 TATTTGTAAA GGTTTCTGTT TAAAATATTT TAAATTTGCA TATCACAACC CTGTGGTAGG 4860 ATGAATTGTT ACTGTTAACT TTTGAACACG CTATGCGTGG TAATTGTTTA ACGAGCAGAC 4920 ATGAAGAAAA CAGGTTAATC CCAGTGGCTT CTCTAGGGGT AGTTGTATAT GGTTCGCATG 4980 GGTGGATGTG TGTGTGCATG TGACTTTCCA ATGTACTGTA TTGTGGTTTG TTGTTGTTGT 5040 TGCTGTTGTT GTTCATTTTG GTGTTTTTGG TTGCTTTGTA TGATCTTAGC TCTGGCCTAG 5100 GTGGGCTGGG AAGGTCCAGG TCTTTTTCTG TCGTGATGCT GGTGGAAAGG TGACCCCAAT 5160 CATCTGTCCT ATTCTCTGGG ACTATTC 5187 1434 amino acids amino acid single linear protein 10 Met Ala Ser Ala Gly Asn Ala Ala Gly Ala Leu Gly Arg Gln Ala Gly 1 5 10 15 Gly Gly Arg Arg Arg Arg Thr Gly Gly Pro His Arg Ala Ala Pro Asp 20 25 30 Arg Asp Tyr Leu His Arg Pro Ser Tyr Cys Asp Ala Ala Phe Ala Leu 35 40 45 Glu Gln Ile Ser Lys Gly Lys Ala Thr Gly Arg Lys Ala Pro Leu Trp 50 55 60 Leu Arg Ala Lys Phe Gln Arg Leu Leu Phe Lys Leu Gly Cys Tyr Ile 65 70 75 80 Gln Lys Asn Cys Gly Lys Phe Leu Val Val Gly Leu Leu Ile Phe Gly 85 90 95 Ala Phe Ala Val Gly Leu Lys Ala Ala Asn Leu Glu Thr Asn Val Glu 100 105 110 Glu Leu Trp Val Glu Val Gly Gly Arg Val Ser Arg Glu Leu Asn Tyr 115 120 125 Thr Arg Gln Lys Ile Gly Glu Glu Ala Met Phe Asn Pro Gln Leu Met 130 135 140 Ile Gln Thr Pro Lys Glu Glu Gly Ala Asn Val Leu Thr Thr Glu Ala 145 150 155 160 Leu Leu Gln His Leu Asp Ser Ala Leu Gln Ala Ser Arg Val His Val 165 170 175 Tyr Met Tyr Asn Arg Gln Trp Lys Leu Glu His Leu Cys Tyr Lys Ser 180 185 190 Gly Glu Leu Ile Thr Glu Thr Gly Tyr Met Asp Gln Ile Ile Glu Tyr 195 200 205 Leu Tyr Pro Cys Leu Ile Ile Thr Pro Leu Asp Cys Phe Trp Glu Gly 210 215 220 Ala Lys Leu Gln Ser Gly Thr Ala Tyr Leu Leu Gly Lys Pro Pro Leu 225 230 235 240 Arg Trp Thr Asn Phe Asp Pro Leu Glu Phe Leu Glu Glu Leu Lys Lys 245 250 255 Ile Asn Tyr Gln Val Asp Ser Trp Glu Glu Met Leu Asn Lys Ala Glu 260 265 270 Val Gly His Gly Tyr Met Asp Arg Pro Cys Leu Asn Pro Ala Asp Pro 275 280 285 Asp Cys Pro Ala Thr Ala Pro Asn Lys Asn Ser Thr Lys Pro Leu Asp 290 295 300 Val Ala Leu Val Leu Asn Gly Gly Cys Gln Gly Leu Ser Arg Lys Tyr 305 310 315 320 Met His Trp Gln Glu Glu Leu Ile Val Gly Gly Thr Val Lys Asn Ala 325 330 335 Thr Gly Lys Leu Val Ser Ala His Ala Leu Gln Thr Met Phe Gln Leu 340 345 350 Met Thr Pro Lys Gln Met Tyr Glu His Phe Arg Gly Tyr Asp Tyr Val 355 360 365 Ser His Ile Asn Trp Asn Glu Asp Arg Ala Ala Ala Ile Leu Glu Ala 370 375 380 Trp Gln Arg Thr Tyr Val Glu Val Val His Gln Ser Val Ala Pro Asn 385 390 395 400 Ser Thr Gln Lys Val Leu Pro Phe Thr Thr Thr Thr Leu Asp Asp Ile 405 410 415 Leu Lys Ser Phe Ser Asp Val Ser Val Ile Arg Val Ala Ser Gly Tyr 420 425 430 Leu Leu Met Leu Ala Tyr Ala Cys Leu Thr Met Leu Arg Trp Asp Cys 435 440 445 Ser Lys Ser Gln Gly Ala Val Gly Leu Ala Gly Val Leu Leu Val Ala 450 455 460 Leu Ser Val Ala Ala Gly Leu Gly Leu Cys Ser Leu Ile Gly Ile Ser 465 470 475 480 Phe Asn Ala Ala Thr Thr Gln Val Leu Pro Phe Leu Ala Leu Gly Val 485 490 495 Gly Val Asp Asp Val Phe Leu Leu Ala His Ala Phe Ser Glu Thr Gly 500 505 510 Gln Asn Lys Arg Ile Pro Phe Glu Asp Arg Thr Gly Glu Cys Leu Lys 515 520 525 Arg Thr Gly Ala Ser Val Ala Leu Thr Ser Ile Ser Asn Val Thr Ala 530 535 540 Phe Phe Met Ala Ala Leu Ile Pro Ile Pro Ala Leu Arg Ala Phe Ser 545 550 555 560 Leu Gln Ala Ala Val Val Val Val Phe Asn Phe Ala Met Val Leu Leu 565 570 575 Ile Phe Pro Ala Ile Leu Ser Met Asp Leu Tyr Arg Arg Glu Asp Arg 580 585 590 Arg Leu Asp Ile Phe Cys Cys Phe Thr Ser Pro Cys Val Ser Arg Val 595 600 605 Ile Gln Val Glu Pro Gln Ala Tyr Thr Glu Pro His Ser Asn Thr Arg 610 615 620 Tyr Ser Pro Pro Pro Pro Tyr Thr Ser His Ser Phe Ala His Glu Thr 625 630 635 640 His Ile Thr Met Gln Ser Thr Val Gln Leu Arg Thr Glu Tyr Asp Pro 645 650 655 His Thr His Val Tyr Tyr Thr Thr Ala Glu Pro Arg Ser Glu Ile Ser 660 665 670 Val Gln Pro Val Thr Val Thr Gln Asp Asn Leu Ser Cys Gln Ser Pro 675 680 685 Glu Ser Thr Ser Ser Thr Arg Asp Leu Leu Ser Gln Phe Ser Asp Ser 690 695 700 Ser Leu His Cys Leu Glu Pro Pro Cys Thr Lys Trp Thr Leu Ser Ser 705 710 715 720 Phe Ala Glu Lys His Tyr Ala Pro Phe Leu Leu Lys Pro Lys Ala Lys 725 730 735 Val Val Val Ile Leu Leu Phe Leu Gly Leu Leu Gly Val Ser Leu Tyr 740 745 750 Gly Thr Thr Arg Val Arg Asp Gly Leu Asp Leu Thr Asp Ile Val Pro 755 760 765 Arg Glu Thr Arg Glu Tyr Asp Phe Ile Ala Ala Gln Phe Lys Tyr Phe 770 775 780 Ser Phe Tyr Asn Met Tyr Ile Val Thr Gln Lys Ala Asp Tyr Pro Asn 785 790 795 800 Ile Gln His Leu Leu Tyr Asp Leu His Lys Ser Phe Ser Asn Val Lys 805 810 815 Tyr Val Met Leu Glu Glu Asn Lys Gln Leu Pro Gln Met Trp Leu His 820 825 830 Tyr Phe Arg Asp Trp Leu Gln Gly Leu Gln Asp Ala Phe Asp Ser Asp 835 840 845 Trp Glu Thr Gly Arg Ile Met Pro Asn Asn Tyr Lys Asn Gly Ser Asp 850 855 860 Asp Gly Val Leu Ala Tyr Lys Leu Leu Val Gln Thr Gly Ser Arg Asp 865 870 875 880 Lys Pro Ile Asp Ile Ser Gln Leu Thr Lys Gln Arg Leu Val Asp Ala 885 890 895 Asp Gly Ile Ile Asn Pro Ser Ala Phe Tyr Ile Tyr Leu Thr Ala Trp 900 905 910 Val Ser Asn Asp Pro Val Ala Tyr Ala Ala Ser Gln Ala Asn Ile Arg 915 920 925 Pro His Arg Pro Glu Trp Val His Asp Lys Ala Asp Tyr Met Pro Glu 930 935 940 Thr Arg Leu Arg Ile Pro Ala Ala Glu Pro Ile Glu Tyr Ala Gln Phe 945 950 955 960 Pro Phe Tyr Leu Asn Gly Leu Arg Asp Thr Ser Asp Phe Val Glu Ala 965 970 975 Ile Glu Lys Val Arg Val Ile Cys Asn Asn Tyr Thr Ser Leu Gly Leu 980 985 990 Ser Ser Tyr Pro Asn Gly Tyr Pro Phe Leu Phe Trp Glu Gln Tyr Ile 995 1000 1005 Ser Leu Arg His Trp Leu Leu Leu Ser Ile Ser Val Val Leu Ala Cys 1010 1015 1020 Thr Phe Leu Val Cys Ala Val Phe Leu Leu Asn Pro Trp Thr Ala Gly 1025 1030 1035 1040 Ile Ile Val Met Val Leu Ala Leu Met Thr Val Glu Leu Phe Gly Met 1045 1050 1055 Met Gly Leu Ile Gly Ile Lys Leu Ser Ala Val Pro Val Val Ile Leu 1060 1065 1070 Ile Ala Ser Val Gly Ile Gly Val Glu Phe Thr Val His Val Ala Leu 1075 1080 1085 Ala Phe Leu Thr Ala Ile Gly Asp Lys Asn His Arg Ala Met Leu Ala 1090 1095 1100 Leu Glu His Met Phe Ala Pro Val Leu Asp Gly Ala Val Ser Thr Leu 1105 1110 1115 1120 Leu Gly Val Leu Met Leu Ala Gly Ser Glu Phe Asp Phe Ile Val Arg 1125 1130 1135 Tyr Phe Phe Ala Val Leu Ala Ile Leu Thr Val Leu Gly Val Leu Asn 1140 1145 1150 Gly Leu Val Leu Leu Pro Val Leu Leu Ser Phe Phe Gly Pro Cys Pro 1155 1160 1165 Glu Val Ser Pro Ala Asn Gly Leu Asn Arg Leu Pro Thr Pro Ser Pro 1170 1175 1180 Glu Pro Pro Pro Ser Val Val Arg Phe Ala Val Pro Pro Gly His Thr 1185 1190 1195 1200 Asn Asn Gly Ser Asp Ser Ser Asp Ser Glu Tyr Ser Ser Gln Thr Thr 1205 1210 1215 Val Ser Gly Ile Ser Glu Glu Leu Arg Gln Tyr Glu Ala Gln Gln Gly 1220 1225 1230 Ala Gly Gly Pro Ala His Gln Val Ile Val Glu Ala Thr Glu Asn Pro 1235 1240 1245 Val Phe Ala Arg Ser Thr Val Val His Pro Asp Ser Arg His Gln Pro 1250 1255 1260 Pro Leu Thr Pro Arg Gln Gln Pro His Leu Asp Ser Gly Ser Leu Ser 1265 1270 1275 1280 Pro Gly Arg Gln Gly Gln Gln Pro Arg Arg Asp Pro Pro Arg Glu Gly 1285 1290 1295 Leu Arg Pro Pro Pro Tyr Arg Pro Arg Arg Asp Ala Phe Glu Ile Ser 1300 1305 1310 Thr Glu Gly His Ser Gly Pro Ser Asn Arg Asp Arg Ser Gly Pro Arg 1315 1320 1325 Gly Ala Arg Ser His Asn Pro Arg Asn Pro Thr Ser Thr Ala Met Gly 1330 1335 1340 Ser Ser Val Pro Ser Tyr Cys Gln Pro Ile Thr Thr Val Thr Ala Ser 1345 1350 1355 1360 Ala Ser Val Thr Val Ala Val His Pro Pro Pro Gly Pro Gly Arg Asn 1365 1370 1375 Pro Arg Gly Gly Pro Cys Pro Gly Tyr Glu Ser Tyr Pro Glu Thr Asp 1380 1385 1390 His Gly Val Phe Glu Asp Pro His Val Pro Phe His Val Arg Cys Glu 1395 1400 1405 Arg Arg Asp Ser Lys Val Glu Val Ile Glu Leu Gln Asp Val Glu Cys 1410 1415 1420 Glu Glu Arg Pro Trp Gly Ser Ser Ser Asn 1425 1430 11 amino acids amino acid single linear peptide 11 Ile Ile Thr Pro Leu Asp Cys Phe Trp Glu Gly 1 5 10 5 amino acids amino acid single linear peptide 12 Leu Ile Val Gly Gly 1 5 7 amino acids amino acid single linear peptide 13 Pro Phe Phe Trp Glu Gln Tyr 1 5 28 base pairs nucleic acid single linear other nucleic acid /desc = “primer” 14 GGACGAATTC AARGTNCAYC ARYTNTGG 28 26 base pairs nucleic acid single linear other nucleic acid /desc = “primer” 15 GGACGAATTC CYTCCCARAA RCANTC 26 27 base pairs nucleic acid single linear other nucleic acid /desc = “primer” 16 GGACGAATTC YTNGANTGYT TYTGGGA 27 31 base pairs nucleic acid single linear other nucleic acid /desc = “primer” 17 CATACCAGCC AAGCTTGTCN GGCCARTGCA T 31 5288 base pairs nucleic acid single linear cDNA 18 GAATTCCGGG GACCGCAAGG AGTGCCGCGG AAGCGCCCGA AGGACAGGCT CGCTCGGCGC 60 GCCGGCTCTC GCTCTTCCGC GAACTGGATG TGGGCAGCGG CGGCCGCAGA GACCTCGGGA 120 CCCCCGCGCA ATGTGGCAAT GGAAGGCGCA GGGTCTGACT CCCCGGCAGC GGCCGCGGCC 180 GCAGCGGCAG CAGCGCCCGC CGTGTGAGCA GCAGCAGCGG CTGGTCTGTC AACCGGAGCC 240 CGAGCCCGAG CAGCCTGCGG CCAGCAGCGT CCTCGCAAGC CGAGCGCCCA GGCGCGCCAG 300 GAGCCCGCAG CAGCGGCAGC AGCGCGCCGG GCCGCCCGGG AAGCCTCCGT CCCCGCGGCG 360 GCGGCGGCGG CGGCGGCGGC AACATGGCCT CGGCTGGTAA CGCCGCCGAG CCCCAGGACC 420 GCGGCGGCGG CGGCAGCGGC TGTATCGGTG CCCCGGGACG GCCGGCTGGA GGCGGGAGGC 480 GCAGACGGAC GGGGGGGCTG CGCCGTGCTG CCGCGCCGGA CCGGGACTAT CTGCACCGGC 540 CCAGCTACTG CGACGCCGCC TTCGCTCTGG AGCAGATTTC CAAGGGGAAG GCTACTGGCC 600 GGAAAGCGCC ACTGTGGCTG AGAGCGAAGT TTCAGAGACT CTTATTTAAA CTGGGTTGTT 660 ACATTCAAAA AAACTGCGGC AAGTTCTTGG TTGTGGGCCT CCTCATATTT GGGGCCTTCG 720 CGGTGGGATT AAAAGCAGCG AACCTCGAGA CCAACGTGGA GGAGCTGTGG GTGGAAGTTG 780 GAGGACGAGT AAGTCGTGAA TTAAATTATA CTCGCCAGAA GATTGGAGAA GAGGCTATGT 840 TTAATCCTCA ACTCATGATA CAGACCCCTA AAGAAGAAGG TGCTAATGTC CTGACCACAG 900 AAGCGCTCCT ACAACACCTG GACTCGGCAC TCCAGGCCAG CCGTGTCCAT GTATACATGT 960 ACAACAGGCA GTGGAAATTG GAACATTTGT GTTACAAATC AGGAGAGCTT ATCACAGAAA 1020 CAGGTTACAT GGATCAGATA ATAGAATATC TTTACCCTTG TTTGATTATT ACACCTTTGG 1080 ACTGCTTCTG GGAAGGGGCG AAATTACAGT CTGGGACAGC ATACCTCCTA GGTAAACCTC 1140 CTTTGCGGTG GACAAACTTC GACCCTTTGG AATTCCTGGA AGAGTTAAAG AAAATAAACT 1200 ATCAAGTGGA CAGCTGGGAG GAAATGCTGA ATAAGGCTGA GGTTGGTCAT GGTTACATGG 1260 ACCGCCCCTG CCTCAATCCG GCCGATCCAG ACTGCCCCGC CACAGCCCCC AACAAAAATT 1320 CAACCAAACC TCTTGATATG GCCCTTGTTT TGAATGGTGG ATGTCATGGC TTATCCAGAA 1380 AGTATATGCA CTGGCAGGAG GAGTTGATTG TGGGTGGCAC AGTCAAGAAC AGCACTGGAA 1440 AACTCGTCAG CGCCCATGCC CTGCAGACCA TGTTCCAGTT AATGACTCCC AAGCAAATGT 1500 ACGAGCACTT CAAGGGGTAC GAGTATGTCT CACACATCAA CTGGAACGAG GACAAAGCGG 1560 CAGCCATCCT GGAGGCCTGG CAGAGGACAT ATGTGGAGGT GGTTCATCAG AGTGTCGCAC 1620 AGAACTCCAC TCAAAAGGTG CTTTCCTTCA CCACCACGAC CCTGGACGAC ATCCTGAAAT 1680 CCTTCTCTGA CGTCAGTGTC ATCCGCGTGG CCAGCGGCTA CTTACTCATG CTCGCCTATG 1740 CCTGTCTAAC CATGCTGCGC TGGGACTGCT CCAAGTCCCA GGGTGCCGTG GGGCTGGCTG 1800 GCGTCCTGCT GGTTGCACTG TCAGTGGCTG CAGGACTGGG CCTGTGCTCA TTGATCGGAA 1860 TTTCCTTTAA CGCTGCAACA ACTCAGGTTT TGCCATTTCT CGCTCTTGGT GTTGGTGTGG 1920 ATGATGTTTT TCTTCTGGCC CACGCCTTCA GTGAAACAGG ACAGAATAAA AGAATCCCTT 1980 TTGAGGACAG GACCGGGGAG TGCCTGAAGC GCACAGGAGC CAGCGTGGCC CTCACGTCCA 2040 TCAGCAATGT CACAGCCTTC TTCATGGCCG CGTTAATCCC AATTCCCGCT CTGCGGGCGT 2100 TCTCCCTCCA GGCAGCGGTA GTAGTGGTGT TCAATTTTGC CATGGTTCTG CTCATTTTTC 2160 CTGCAATTCT CAGCATGGAT TTATATCGAC GCGAGGACAG GAGACTGGAT ATTTTCTGCT 2220 GTTTTACAAG CCCCTGCGTC AGCAGAGTGA TTCAGGTTGA ACCTCAGGCC TACACCGACA 2280 CACACGACAA TACCCGCTAC AGCCCCCCAC CTCCCTACAG CAGCCACAGC TTTGCCCATG 2340 AAACGCAGAT TACCATGCAG TCCACTGTCC AGCTCCGCAC GGAGTACGAC CCCCACACGC 2400 ACGTGTACTA CACCACCGCT GAGCCGCGCT CCGAGATCTC TGTGCAGCCC GTCACCGTGA 2460 CACAGGACAC CCTCAGCTGC CAGAGCCCAG AGAGCACCAG CTCCACAAGG GACCTGCTCT 2520 CCCAGTTCTC CGACTCCAGC CTCCACTGCC TCGAGCCCCC CTGTACGAAG TGGACACTCT 2580 CATCTTTTGC TGAGAAGCAC TATGCTCCTT TCCTCTTGAA ACCAAAAGCC AAGGTAGTGG 2640 TGATCTTCCT TTTTCTGGGC TTGCTGGGGG TCAGCCTTTA TGGCACCACC CGAGTGAGAG 2700 ACGGGCTGGA CCTTACGGAC ATTGTACCTC GGGAAACCAG AGAATATGAC TTTATTGCTG 2760 CACAATTCAA ATACTTTTCT TTCTACAACA TGTATATAGT CACCCAGAAA GCAGACTACC 2820 CGAATATCCA GCACTTACTT TACGACCTAC ACAGGAGTTT CAGTAACGTG AAGTATGTCA 2880 TGTTGGAAGA AAACAAACAG CTTCCCAAAA TGTGGCTGCA CTACTTCAGA GACTGGCTTC 2940 AGGGACTTCA GGATGCATTT GACAGTGACT GGGAAACCGG GAAAATCATG CCAAACAATT 3000 ACAAGAATGG ATCAGACGAT GGAGTCCTTG CCTACAAACT CCTGGTGCAA ACCGGCAGCC 3060 GCGATAAGCC CATCGACATC AGCCAGTTGA CTAAACAGCG TCTGGTGGAT GCAGATGGCA 3120 TCATTAATCC CAGCGCTTTC TACATCTACC TGACGGCTTG GGTCAGCAAC GACCCCGTCG 3180 CGTATGCTGC CTCCCAGGCC AACATCCGGC CACACCGACC AGAATGGGTC CACGACAAAG 3240 CCGACTACAT GCCTGAAACA AGGCTGAGAA TCCCGGCAGC AGAGCCCATC GAGTATGCCC 3300 AGTTCCCTTT CTACCTCAAC GGGTTGCGGG ACACCTCAGA CTTTGTGGAG GCAATTGAAA 3360 AAGTAAGGAC CATCTGCAGC AACTATACGA GCCTGGGGCT GTCCAGTTAC CCCAACGGCT 3420 ACCCCTTCCT CTTCTGGGAG CAGTACATCG GCCTCCGCCA CTGGCTGCTG CTGTTCATCA 3480 GCGTGGTGTT GGCCTGCACA TTCCTCGTGT GCGCTGTCTT CCTTCTGAAC CCCTGGACGG 3540 CCGGGATCAT TGTGATGGTC CTGGCGCTGA TGACGGTCGA GCTGTTCGGC ATGATGGGCC 3600 TCATCGGAAT CAAGCTCAGT GCCGTGCCCG TGGTCATCCT GATCGCTTCT GTTGGCATAG 3660 GAGTGGAGTT CACCGTTCAC GTTGCTTTGG CCTTTCTGAC GGCCATCGGC GACAAGAACC 3720 GCAGGGCTGT GCTTGCCCTG GAGCACATGT TTGCACCCGT CCTGGATGGC GCCGTGTCCA 3780 CTCTGCTGGG AGTGCTGATG CTGGCGGGAT CTGAGTTCGA CTTCATTGTC AGGTATTTCT 3840 TTGCTGTGCT GGCGATCCTC ACCATCCTCG GCGTTCTCAA TGGGCTGGTT TTGCTTCCCG 3900 TGCTTTTGTC TTTCTTTGGA CCATATCCTG AGGTGTCTCC AGCCAACGGC TTGAACCGCC 3960 TGCCCACACC CTCCCCTGAG CCACCCCCCA GCGTGGTCCG CTTCGCCATG CCGCCCGGCC 4020 ACACGCACAG CGGGTCTGAT TCCTCCGACT CGGAGTATAG TTCCCAGACG ACAGTGTCAG 4080 GCCTCAGCGA GGAGCTTCGG CACTACGAGG CCCAGCAGGG CGCGGGAGGC CCTGCCCACC 4140 AAGTGATCGT GGAAGCCACA GAAAACCCCG TCTTCGCCCA CTCCACTGTG GTCCATCCCG 4200 AATCCAGGCA TCACCCACCC TCGAACCCGA GACAGCAGCC CCACCTGGAC TCAGGGTCCC 4260 TGCCTCCCGG ACGGCAAGGC CAGCAGCCCC GCAGGGACCC CCCCAGAGAA GGCTTGTGGC 4320 CACCCCTCTA CAGACCGCGC AGAGACGCTT TTGAAATTTC TACTGAAGGG CATTCTGGCC 4380 CTAGCAATAG GGCCCGCTGG GGCCCTCGCG GGGCCCGTTC TCACAACCCT CGGAACCCAG 4440 CGTCCACTGC CATGGGCAGC TCCGTGCCCG GCTACTGCCA GCCCATCACC ACTGTGACGG 4500 CTTCTGCCTC CGTGACTGTC GCCGTGCACC CGCCGCCTGT CCCTGGGCCT GGGCGGAACC 4560 CCCGAGGGGG ACTCTGCCCA GGCTACCCTG AGACTGACCA CGGCCTGTTT GAGGACCCCC 4620 ACGTGCCTTT CCACGTCCGG TGTGAGAGGA GGGATTCGAA GGTGGAAGTC ATTGAGCTGC 4680 AGGACGTGGA ATGCGAGGAG AGGCCCCGGG GAAGCAGCTC CAACTGAGGG TGATTAAAAT 4740 CTGAAGCAAA GAGGCCAAAG ATTGGAAACC CCCCACCCCC ACCTCTTTCC AGAACTGCTT 4800 GAAGAGAACT GGTTGGAGTT ATGGAAAAGA TGCCCTGTGC CAGGACAGCA GTTCATTGTT 4860 ACTGTAACCG ATTGTATTAT TTTGTTAAAT ATTTCTATAA ATATTTAAGA GATGTACACA 4920 TGTGTAATAT AGGAAGGAAG GATGTAAAGT GGTATGATCT GGGGCTTCTC CACTCCTGCC 4980 CCAGAGTGTG GAGGCCACAG TGGGGCCTCT CCGTATTTGT GCATTGGGCT CCGTGCCACA 5040 ACCAAGCTTC ATTAGTCTTA AATTTCAGCA TATGTTGCTG CTGCTTAAAT ATTGTATAAT 5100 TTACTTGTAT AATTCTATGC AAATATTGCT TATGTAATAG GATTATTTTG TAAAGGTTTC 5160 TGTTTAAAAT ATTTTAAATT TGCATATCAC AACCCTGTGG TAGTATGAAA TGTTACTGTT 5220 AACTTTCAAA CACGCTATGC GTGATAATTT TTTTGTTTAA TGAGCAGATA TGAAGAAAGC 5280 CCGGAATT 5288 1447 amino acids amino acid single linear protein 19 Met Ala Ser Ala Gly Asn Ala Ala Glu Pro Gln Asp Arg Gly Gly Gly 1 5 10 15 Gly Ser Gly Cys Ile Gly Ala Pro Gly Arg Pro Ala Gly Gly Gly Arg 20 25 30 Arg Arg Arg Thr Gly Gly Leu Arg Arg Ala Ala Ala Pro Asp Arg Asp 35 40 45 Tyr Leu His Arg Pro Ser Tyr Cys Asp Ala Ala Phe Ala Leu Glu Gln 50 55 60 Ile Ser Lys Gly Lys Ala Thr Gly Arg Lys Ala Pro Leu Trp Leu Arg 65 70 75 80 Ala Lys Phe Gln Arg Leu Leu Phe Lys Leu Gly Cys Tyr Ile Gln Lys 85 90 95 Asn Cys Gly Lys Phe Leu Val Val Gly Leu Leu Ile Phe Gly Ala Phe 100 105 110 Ala Val Gly Leu Lys Ala Ala Asn Leu Glu Thr Asn Val Glu Glu Leu 115 120 125 Trp Val Glu Val Gly Gly Arg Val Ser Arg Glu Leu Asn Tyr Thr Arg 130 135 140 Gln Lys Ile Gly Glu Glu Ala Met Phe Asn Pro Gln Leu Met Ile Gln 145 150 155 160 Thr Pro Lys Glu Glu Gly Ala Asn Val Leu Thr Thr Glu Ala Leu Leu 165 170 175 Gln His Leu Asp Ser Ala Leu Gln Ala Ser Arg Val His Val Tyr Met 180 185 190 Tyr Asn Arg Gln Trp Lys Leu Glu His Leu Cys Tyr Lys Ser Gly Glu 195 200 205 Leu Ile Thr Glu Thr Gly Tyr Met Asp Gln Ile Ile Glu Tyr Leu Tyr 210 215 220 Pro Cys Leu Ile Ile Thr Pro Leu Asp Cys Phe Trp Glu Gly Ala Lys 225 230 235 240 Leu Gln Ser Gly Thr Ala Tyr Leu Leu Gly Lys Pro Pro Leu Arg Trp 245 250 255 Thr Asn Phe Asp Pro Leu Glu Phe Leu Glu Glu Leu Lys Lys Ile Asn 260 265 270 Tyr Gln Val Asp Ser Trp Glu Glu Met Leu Asn Lys Ala Glu Val Gly 275 280 285 His Gly Tyr Met Asp Arg Pro Cys Leu Asn Pro Ala Asp Pro Asp Cys 290 295 300 Pro Ala Thr Ala Pro Asn Lys Asn Ser Thr Lys Pro Leu Asp Met Ala 305 310 315 320 Leu Val Leu Asn Gly Gly Cys His Gly Leu Ser Arg Lys Tyr Met His 325 330 335 Trp Gln Glu Glu Leu Ile Val Gly Gly Thr Val Lys Asn Ser Thr Gly 340 345 350 Lys Leu Val Ser Ala His Ala Leu Gln Thr Met Phe Gln Leu Met Thr 355 360 365 Pro Lys Gln Met Tyr Glu His Phe Lys Gly Tyr Glu Tyr Val Ser His 370 375 380 Ile Asn Trp Asn Glu Asp Lys Ala Ala Ala Ile Leu Glu Ala Trp Gln 385 390 395 400 Arg Thr Tyr Val Glu Val Val His Gln Ser Val Ala Gln Asn Ser Thr 405 410 415 Gln Lys Val Leu Ser Phe Thr Thr Thr Thr Leu Asp Asp Ile Leu Lys 420 425 430 Ser Phe Ser Asp Val Ser Val Ile Arg Val Ala Ser Gly Tyr Leu Leu 435 440 445 Met Leu Ala Tyr Ala Cys Leu Thr Met Leu Arg Trp Asp Cys Ser Lys 450 455 460 Ser Gln Gly Ala Val Gly Leu Ala Gly Val Leu Leu Val Ala Leu Ser 465 470 475 480 Val Ala Ala Gly Leu Gly Leu Cys Ser Leu Ile Gly Ile Ser Phe Asn 485 490 495 Ala Ala Thr Thr Gln Val Leu Pro Phe Leu Ala Leu Gly Val Gly Val 500 505 510 Asp Asp Val Phe Leu Leu Ala His Ala Phe Ser Glu Thr Gly Gln Asn 515 520 525 Lys Arg Ile Pro Phe Glu Asp Arg Thr Gly Glu Cys Leu Lys Arg Thr 530 535 540 Gly Ala Ser Val Ala Leu Thr Ser Ile Ser Asn Val Thr Ala Phe Phe 545 550 555 560 Met Ala Ala Leu Ile Pro Ile Pro Ala Leu Arg Ala Phe Ser Leu Gln 565 570 575 Ala Ala Val Val Val Val Phe Asn Phe Ala Met Val Leu Leu Ile Phe 580 585 590 Pro Ala Ile Leu Ser Met Asp Leu Tyr Arg Arg Glu Asp Arg Arg Leu 595 600 605 Asp Ile Phe Cys Cys Phe Thr Ser Pro Cys Val Ser Arg Val Ile Gln 610 615 620 Val Glu Pro Gln Ala Tyr Thr Asp Thr His Asp Asn Thr Arg Tyr Ser 625 630 635 640 Pro Pro Pro Pro Tyr Ser Ser His Ser Phe Ala His Glu Thr Gln Ile 645 650 655 Thr Met Gln Ser Thr Val Gln Leu Arg Thr Glu Tyr Asp Pro His Thr 660 665 670 His Val Tyr Tyr Thr Thr Ala Glu Pro Arg Ser Glu Ile Ser Val Gln 675 680 685 Pro Val Thr Val Thr Gln Asp Thr Leu Ser Cys Gln Ser Pro Glu Ser 690 695 700 Thr Ser Ser Thr Arg Asp Leu Leu Ser Gln Phe Ser Asp Ser Ser Leu 705 710 715 720 His Cys Leu Glu Pro Pro Cys Thr Lys Trp Thr Leu Ser Ser Phe Ala 725 730 735 Glu Lys His Tyr Ala Pro Phe Leu Leu Lys Pro Lys Ala Lys Val Val 740 745 750 Val Ile Phe Leu Phe Leu Gly Leu Leu Gly Val Ser Leu Tyr Gly Thr 755 760 765 Thr Arg Val Arg Asp Gly Leu Asp Leu Thr Asp Ile Val Pro Arg Glu 770 775 780 Thr Arg Glu Tyr Asp Phe Ile Ala Ala Gln Phe Lys Tyr Phe Ser Phe 785 790 795 800 Tyr Asn Met Tyr Ile Val Thr Gln Lys Ala Asp Tyr Pro Asn Ile Gln 805 810 815 His Leu Leu Tyr Asp Leu His Arg Ser Phe Ser Asn Val Lys Tyr Val 820 825 830 Met Leu Glu Glu Asn Lys Gln Leu Pro Lys Met Trp Leu His Tyr Phe 835 840 845 Arg Asp Trp Leu Gln Gly Leu Gln Asp Ala Phe Asp Ser Asp Trp Glu 850 855 860 Thr Gly Lys Ile Met Pro Asn Asn Tyr Lys Asn Gly Ser Asp Asp Gly 865 870 875 880 Val Leu Ala Tyr Lys Leu Leu Val Gln Thr Gly Ser Arg Asp Lys Pro 885 890 895 Ile Asp Ile Ser Gln Leu Thr Lys Gln Arg Leu Val Asp Ala Asp Gly 900 905 910 Ile Ile Asn Pro Ser Ala Phe Tyr Ile Tyr Leu Thr Ala Trp Val Ser 915 920 925 Asn Asp Pro Val Ala Tyr Ala Ala Ser Gln Ala Asn Ile Arg Pro His 930 935 940 Arg Pro Glu Trp Val His Asp Lys Ala Asp Tyr Met Pro Glu Thr Arg 945 950 955 960 Leu Arg Ile Pro Ala Ala Glu Pro Ile Glu Tyr Ala Gln Phe Pro Phe 965 970 975 Tyr Leu Asn Gly Leu Arg Asp Thr Ser Asp Phe Val Glu Ala Ile Glu 980 985 990 Lys Val Arg Thr Ile Cys Ser Asn Tyr Thr Ser Leu Gly Leu Ser Ser 995 1000 1005 Tyr Pro Asn Gly Tyr Pro Phe Leu Phe Trp Glu Gln Tyr Ile Gly Leu 1010 1015 1020 Arg His Trp Leu Leu Leu Phe Ile Ser Val Val Leu Ala Cys Thr Phe 1025 1030 1035 1040 Leu Val Cys Ala Val Phe Leu Leu Asn Pro Trp Thr Ala Gly Ile Ile 1045 1050 1055 Val Met Val Leu Ala Leu Met Thr Val Glu Leu Phe Gly Met Met Gly 1060 1065 1070 Leu Ile Gly Ile Lys Leu Ser Ala Val Pro Val Val Ile Leu Ile Ala 1075 1080 1085 Ser Val Gly Ile Gly Val Glu Phe Thr Val His Val Ala Leu Ala Phe 1090 1095 1100 Leu Thr Ala Ile Gly Asp Lys Asn Arg Arg Ala Val Leu Ala Leu Glu 1105 1110 1115 1120 His Met Phe Ala Pro Val Leu Asp Gly Ala Val Ser Thr Leu Leu Gly 1125 1130 1135 Val Leu Met Leu Ala Gly Ser Glu Phe Asp Phe Ile Val Arg Tyr Phe 1140 1145 1150 Phe Ala Val Leu Ala Ile Leu Thr Ile Leu Gly Val Leu Asn Gly Leu 1155 1160 1165 Val Leu Leu Pro Val Leu Leu Ser Phe Phe Gly Pro Tyr Pro Glu Val 1170 1175 1180 Ser Pro Ala Asn Gly Leu Asn Arg Leu Pro Thr Pro Ser Pro Glu Pro 1185 1190 1195 1200 Pro Pro Ser Val Val Arg Phe Ala Met Pro Pro Gly His Thr His Ser 1205 1210 1215 Gly Ser Asp Ser Ser Asp Ser Glu Tyr Ser Ser Gln Thr Thr Val Ser 1220 1225 1230 Gly Leu Ser Glu Glu Leu Arg His Tyr Glu Ala Gln Gln Gly Ala Gly 1235 1240 1245 Gly Pro Ala His Gln Val Ile Val Glu Ala Thr Glu Asn Pro Val Phe 1250 1255 1260 Ala His Ser Thr Val Val His Pro Glu Ser Arg His His Pro Pro Ser 1265 1270 1275 1280 Asn Pro Arg Gln Gln Pro His Leu Asp Ser Gly Ser Leu Pro Pro Gly 1285 1290 1295 Arg Gln Gly Gln Gln Pro Arg Arg Asp Pro Pro Arg Glu Gly Leu Trp 1300 1305 1310 Pro Pro Leu Tyr Arg Pro Arg Arg Asp Ala Phe Glu Ile Ser Thr Glu 1315 1320 1325 Gly His Ser Gly Pro Ser Asn Arg Ala Arg Trp Gly Pro Arg Gly Ala 1330 1335 1340 Arg Ser His Asn Pro Arg Asn Pro Ala Ser Thr Ala Met Gly Ser Ser 1345 1350 1355 1360 Val Pro Gly Tyr Cys Gln Pro Ile Thr Thr Val Thr Ala Ser Ala Ser 1365 1370 1375 Val Thr Val Ala Val His Pro Pro Pro Val Pro Gly Pro Gly Arg Asn 1380 1385 1390 Pro Arg Gly Gly Leu Cys Pro Gly Tyr Pro Glu Thr Asp His Gly Leu 1395 1400 1405 Phe Glu Asp Pro His Val Pro Phe His Val Arg Cys Glu Arg Arg Asp 1410 1415 1420 Ser Lys Val Glu Val Ile Glu Leu Gln Asp Val Glu Cys Glu Glu Arg 1425 1430 1435 1440 Pro Arg Gly Ser Ser Ser Asn 1445 

What is claimed is:
 1. A DNA sequence other than present in a chromosome encoding a patched gene other than the Drosophila patched gene or fragment thereof of at least about 12 bp different from the sequence of the Drosophila patched gene.
 2. A DNA sequence according to claim 1, wherein said patched gene is a mammalian gene.
 3. A DNA sequence according to claim 1 for human, mouse, mosquito, butterfly or beetle patched gene.
 4. A DNA sequence according to claim 3, wherein said DNA sequence is a human sequence.
 5. A DNA sequence according to claim 4, wherein said DNA sequence is a mouse sequence.
 6. A DNA sequence according to claim 1, wherein said DNA sequence is a fragment of at least about 18 bp.
 7. A DNA sequence according to claim 1 joined to a DNA sequence comprising a restriction enzyme recognition sequence.
 8. An expression cassette comprising a transcriptional initiation region functional in an expression host, a DNA sequence according to claim 1 under the transcriptional regulation of said transcriptional initiation region, and a transcriptional termination region functional in said expression host.
 9. An expression cassette according to claim 8, wherein said transcriptional initiation region is heterologous to said DNA sequence according to claim
 1. 10. An expression cassette according to claim 8, wherein said transcriptional initiation region is homologous to said DNA sequence according to claim 1 and includes the enhancer region.
 11. A cell comprising an expression cassette according to claim 8 as part of an extrachromosomal element or integrated into the genome of a host cell as a result of introduction of said expression cassette into said host cell and the cellular progeny of said host cell.
 12. A cell according to claim 11, further comprising the patched protein in the cellular membrane of said cell.
 13. A cell according to claim 11, wherein said patched protein is a mouse patched protein.
 14. A cell according to claim 11, wherein said patched gene is a human patched protein.
 15. A cell according to claim 11, wherein said transcriptional initiation region is a Drosophila patched gene transcriptional initiation region comprising the promoter and enhancer joined to a heterologous gene.
 16. A cell comprising an expression cassette comprising a transcriptional initiation region functional in an expression host, said transcriptional initiation region consisting of a 5′ non-coding region regulating the transcription of patched protein comprising the promoter and enhancer, a marker gene, and a transcriptional termination region, as part of an extrachromosomal element or integrated into the genome of a host cell as a result of introduction of said expression cassette into said host, and the cellular progeny thereof.
 17. A cell according to claim 16, wherein said transcriptional initiation region is the Drosophila region.
 18. A method for following embryonic development employing the patched protein in an embryo, said method comprising: integrating an expression cassette comprising a transcriptional initiation region functional in embryonic host cells, said transcriptional initiation region consisting of a 5′ non-coding region regulating the transcription of patched protein, a marker gene, and a transcriptional termination region, wherein said embryonic host cells are capable of developing into a fetus; growing said embryonic host cells, whereby proliferation and differentiation occur; and locating cells comprising expression of the patched protein by means of expression of said marker gene.
 19. A method for producing patched protein, said method comprising: growing a cell according to claim 11, whereby said patched protein is expressed; and isolating said patched protein free of other proteins.
 20. A method for screening candidate compounds for binding affinity to the patched protein, said method comprising: combining said candidate protein with a vertebrate or invertebrate cell comprising said patched protein in the membrane of said cell and an expression cassette comprising a transcriptional initiation region functional in said cell, a DNA sequence according to claim 1 comprising the entire coding sequence under the transcriptional regulation of said transcriptional initiation region, and a transcriptional termination region functional in said cell, expressing said patched protein in said cell; and assaying for the binding of said candidate compound to said patched protein.
 21. A method for screening candidate compounds for agonist activity with the patched protein, said method comprising: combining said candidate protein with a vertebrate or invertebrate cell comprising said patched protein in the membrane of said cell and an expression cassette comprising a transcriptional initiation region functional in an expression host, said transcriptional initiation region consisting of a 5′ non-coding region regulating the transcription of patched protein, a marker gene, and a transcriptional termination region, as part of an extrachromosomal element or integrated into the genome of a host cell; and assaying for the expression of said marker gene.
 22. A monoclonal antibody binding specifically to a patched protein, other than the Drosophila patched protein. 